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Active site architecture of an acetyl xylan esterase indicates a novel cold adaptation strategy

  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Yi Zhang
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China

    Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Hai-Tao Ding
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai, China
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  • Wen-Xin Jiang
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Xia Zhang
    Affiliations
    Department of Molecular Biology, Qingdao Vland Biotech Inc, Qingdao, China
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  • Hai-Yan Cao
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Jing-Ping Wang
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Chun-Yang Li
    Affiliations
    College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China

    Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China
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  • Feng Huang
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Xi-Ying Zhang
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Xiu-Lan Chen
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Yu-Zhong Zhang
    Correspondence
    For correspondence: Ping-Yi Li; Yu-Zhong Zhang
    Affiliations
    College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China

    Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China

    State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
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  • Ping-Yi Li
    Correspondence
    For correspondence: Ping-Yi Li; Yu-Zhong Zhang
    Affiliations
    State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
Open AccessPublished:May 27, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100841
      SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.

      Keywords

      Abbreviations:

      AcXEs (acetyl xylan esterases), AlAXEase (a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T), CEs (carbohydrate esterases), CpPAH (phenylalanine hydroxylase from Colwellia psychrerythraea 34H), DLS (dynamic light scattering), DpIDH (isocitrate dehydrogenase from Desulfotalea psychrophila), PDB (Protein Data Bank), pNPC2 (p-nitrophenyl acetate), SeMet (selenomethionine), Topt (optimum temperature)
      Acetyl xylan esterases (AcXEs) (EC 3.1.1.72) are a kind of carbohydrate esterases (CEs) that hydrolyze ester bonds to liberate acetic acid from acetylated hemicellulose, typically xylans and xylooligosaccharides (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ). AcXEs are widely distributed in bacteria, fungi, plants, and mammals (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ). Microbial AcXEs are capable of facilitating the access of main-chain depolymerizing enzymes to xylan (
      • Komiya D.
      • Hori A.
      • Ishida T.
      • Igarashi K.
      • Samejima M.
      • Koseki T.
      • Fushinobu S.
      Crystal structure and substrate specificity modification of acetyl xylan esterase from Aspergillus luchuensis.
      ), thereby playing an important role in terrestrial and marine xylan degradation and recycling.
      Based on amino acid similarities, microbial AcXEs fall into nine families, CE families 1 to 7, 12, and 16 (
      • Lombard V.
      • Golaconda Ramulu H.
      • Drula E.
      • Coutinho P.M.
      • Henrissat B.
      The carbohydrate-active enzymes database (CAZy) in 2013.
      ), as well as a novel CE family (the Axe2 family) recently proposed based on the studies of two AcXEs, Axe2 and Cbes-AcXE2 (
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ,
      • Soni S.
      • Sathe S.S.
      • Odaneth A.A.
      • Lali A.M.
      • Chandrayan S.K.
      SGNH hydrolase-type esterase domain containing Cbes-AcXE2: A novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.
      ). Although some AcXEs are found only to deacetylate xylooligosaccharides (
      • Soni S.
      • Sathe S.S.
      • Odaneth A.A.
      • Lali A.M.
      • Chandrayan S.K.
      SGNH hydrolase-type esterase domain containing Cbes-AcXE2: A novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.
      ,
      • Koutaniemi S.
      • van Gool M.P.
      • Juvonen M.
      • Jokela J.
      • Hinz S.W.
      • Schols H.A.
      • Tenkanen M.
      Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation.
      ), most AcXEs are reported to be active on both acetyl xylan and acetylated xylooligosaccharides (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ,
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ). In addition to the hydrolysis of acetylated xylooligosaccharides, many AcXEs are also capable of hydrolyzing other acetylated oligosaccharides/monosaccharides with relatively high activities, especially for the acetylated glucose (
      • Tian Q.
      • Song P.
      • Jiang L.
      • Li S.
      • Huang H.
      A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid.
      ,
      • Park S.H.
      • Yoo W.
      • Lee C.W.
      • Jeong C.S.
      • Shin S.C.
      • Kim H.W.
      • Park H.
      • Kim K.K.
      • Kim T.D.
      • Lee J.H.
      Crystal structure and functional characterization of a cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp.
      ,
      • Razeq F.M.
      • Jurak E.
      • Stogios P.J.
      • Yan R.
      • Tenkanen M.
      • Kabel M.A.
      • Wang W.
      • Master E.R.
      A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans.
      ). Except for those from families CE1, CE4, CE5, and CE7, AcXEs from the other CE families also belong to the SGNH hydrolase subfamily of the GDSL family (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ,
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ). GDSL family esterases are characterized by the nucleophilic serine located in the conserved GDSL motif rather than in the canonical GxSxG motif reported in other serine esterases (
      • Akoh C.C.
      • Lee G.C.
      • Liaw Y.C.
      • Huang T.H.
      • Shaw J.F.
      GDSL family of serine esterases/lipases.
      ). Some of the GDSL enzymes are further classified into the SGNH hydrolase subfamily because of the presence of four strictly conserved active site residues Ser, Gly, Asn, and His in the four conserved blocks I, II, III, and V, respectively (
      • Li J.
      • Derewenda U.
      • Dauter Z.
      • Smith S.
      • Derewenda Z.S.
      Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.
      ,
      • Molgaard A.
      • Kauppinen S.
      • Larsen S.
      Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases.
      ). The SGNH hydrolase adopts a three-layered αβα sandwich-like fold (
      • Akoh C.C.
      • Lee G.C.
      • Liaw Y.C.
      • Huang T.H.
      • Shaw J.F.
      GDSL family of serine esterases/lipases.
      ,
      • Lescic Asler I.
      • Stefanic Z.
      • Marsavelski A.
      • Vianello R.
      • Kojic-Prodic B.
      Catalytic dyad in the SGNH hydrolase superfamily: In-depth insight into structural parameters tuning the catalytic process of extracellular lipase from Streptomyces rimosus.
      ). Among the SGNH-type AcXEs, only structures of the AcXEs from families CE2, CE3, and CE6, and the Axe2 family have been reported. Structural analyses reveal that AcXEs from CE3, CE6, and the Axe2 family possess a typical SGNH hydrolase fold with a standard Ser-His-Asp catalytic triad (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ,
      • Correia M.A.S.
      • Prates J.A.M.
      • Bras J.
      • Fontes C.M.G.A.
      • Newman J.A.
      • Lewis R.J.
      • Gilbert H.J.
      • Flint J.E.
      Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition.
      ,
      • Lansky S.
      • Alalouf O.
      • Solomon H.V.
      • Alhassid A.
      • Govada L.
      • Chayen N.E.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
      ), whereas members from CE2 are bidomain enzymes, containing an N-terminal β-sheet domain and a C-terminal SGNH-hydrolase domain, and generally have a Ser-His catalytic diad (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ,
      • Till M.
      • Goldstone D.C.
      • Attwood G.T.
      • Moon C.D.
      • Kelly W.J.
      • Arcus V.L.
      Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus.
      ).
      For the SGNH-type AcXEs, while most characterized enzymes are mesophilic or thermophilic, which usually originate from warm terrestrial environments (
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ,
      • Soni S.
      • Sathe S.S.
      • Odaneth A.A.
      • Lali A.M.
      • Chandrayan S.K.
      SGNH hydrolase-type esterase domain containing Cbes-AcXE2: A novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.
      ,
      • Martinez-Martinez I.
      • Navarro-Fernandez J.
      • Daniel Lozada-Ramirez J.
      • Garcia-Carmona F.
      • Sanchez-Ferrer A.
      YesT: A new rhamnogalacturonan acetyl esterase from Bacillus subtilis.
      ), AxeA from the anaerobic rumen fungus Orpinomyces sp. strain PC-2 has been reported to be cold adapted (
      • Blum D.L.
      • Li X.L.
      • Chen H.Z.
      • Ljungdahl L.G.
      Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.
      ). AxeA efficiently deacetylates acetylated xylan at low temperatures of 15 to 30 °C (
      • Blum D.L.
      • Li X.L.
      • Chen H.Z.
      • Ljungdahl L.G.
      Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.
      ). However, owing to the lack of structural information, the underlying mechanisms for the cold adaptation of AcXEs are still unknown by far.
      Cold-adapted enzymes help their source strains to adapt to extremely cold environments. Compared with mesophilic/thermophilic homologs, cold-adapted enzymes display higher catalytic activity at low temperatures because of their more flexible structures (
      • Nagel Z.D.
      • Cun S.
      • Klinman J.P.
      Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases.
      ,
      • Papaleo E.
      • Riccardi L.
      • Villa C.
      • Fantucci P.
      • De Gioia L.
      Flexibility and enzymatic cold-adaptation: A comparative molecular dynamics investigation of the elastase family.
      ). Most cold-adapted enzymes show a global rather than uniform distribution of their flexibility throughout the whole structure, therefore resulting in their low thermostability (
      • Siddiqui K.S.
      • Cavicchioli R.
      Cold-adapted enzymes.
      ). However, some cold-adapted enzymes are also reported to have unusual thermal stability, such as the vibriolysin E495 from an Arctic sea ice bacterium (
      • Xie B.B.
      • Bian F.
      • Chen X.L.
      • He H.L.
      • Guo J.
      • Gao X.
      • Zeng Y.X.
      • Chen B.
      • Zhou B.C.
      • Zhang Y.Z.
      Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and Arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and hydrogen bonding.
      ), the phenylalanine hydroxylase CpPAH from Colwellia psychrerythraea 34H (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ), and the isocitrate dehydrogenase DpIDH from Desulfotalea psychrophila (
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ). Compared with its mesophilic homologs, E495 has similar noncovalent stabilizing interactions but higher flexibility because of the reduction of hydrogen-bond stability in the dynamic structure, suggesting the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation of enzymes (
      • Xie B.B.
      • Bian F.
      • Chen X.L.
      • He H.L.
      • Guo J.
      • Gao X.
      • Zeng Y.X.
      • Chen B.
      • Zhou B.C.
      • Zhang Y.Z.
      Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and Arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and hydrogen bonding.
      ). For CpPAH and DpIDH, both enzymes have local flexibility around their active sites, which leads to their cold adaptation without compromising the global stability of proteins (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ,
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ). Thus, cold-active enzymes may have diverse cold adaption strategies, and the cold adaption mechanisms for other enzymes, especially for the SGNH-type AcXEs, need to be further explored.
      Arcticibacterium luteifluviistationis SM1504T is a cold-adapted bacterium isolated from an Arctic surface seawater sample from King’s Fjord, Svalbard (
      • Li D.D.
      • Peng M.
      • Wang N.
      • Wang X.J.
      • Zhang X.Y.
      • Chen X.L.
      • Su H.N.
      • Zhang Y.Z.
      • Shi M.
      Arcticibacterium luteifluviistationis gen. nov., sp. nov., isolated from Arctic seawater.
      ,
      • Li Y.
      • Guo X.H.
      • Dang Y.R.
      • Sun L.L.
      • Zhang X.Y.
      • Chen X.L.
      • Qin Q.L.
      • Wang P.
      Complete genome sequence of Arcticibacterium luteifluviistationis SM1504T, a cytophagaceae bacterium isolated from Arctic surface seawater.
      ). Here, we identified and characterized a novel SGNH-type AcXE, AlAXEase, from strain SM1504T, which, together with its homologs, represents a new SGNH-type CE family. AlAXEase deacetylated xylooligosaccharides and acetyl xylan. Biochemical characterization showed that AlAXEase is a cold-adapted enzyme with high thermostability. We further solved the crystal structure of AlAXEase to probe the structural basis for its cold adaptation. Structural and enzyme kinetic analyses combined with molecular dynamics (MD) simulations of WT AlAXEase and its mutant E190A at different temperatures revealed that AlAXEase has a long and flexible catalytic loop around its active site that contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32.

      Results

      AlAXEase belongs to a novel SGNH-type CE family

      A gene encoding a GDSL family protein (GenBank Accession No. WP_111370902) was obtained from the genome sequence of the marine cold-adapted bacterium A. luteifluviistationis SM1504T based on gene annotation, which was designated as AlAXEase. AlAXEase is 669 bp in length, encoding a putative lipolytic enzyme of 222 amino acid residues. Based on the SignalP 5.0 prediction, AlAXEase contains an N-terminal signal peptide sequence (14 residues in length).
      AlAXEase shows the highest sequence identity (66%) to an uncharacterized GDSL family protein from Emticicia aquatilis (GenBank Accession No. WP_188769581). Among all the characterized GDSL enzymes, AlAXEase is most closely related to the SGNH-type acetyl xylan esterase Axe2 from Geobacillus stearothermophilus (
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ), with a low sequence identity of 24%, suggesting that AlAXEase is a potential novel SGNH-type CE. To reveal the relationship between AlAXEase and other CEs, a phylogenetic tree was constructed, including AlAXEase and its homologs, Axe2 and its homologs, and characterized enzymes from known SGNH-type CE families 2, 3, 6, 12, and 16 (Fig. 1). The tree showed that AlAXEase and its homologs are clustered as a separate group from all other characterized SGNH-type CEs (Fig. 1). Based on these data, we suggest that AlAXEase and its homologs represent a new SGNH-type CE family.
      Figure thumbnail gr1
      Figure 1Phylogenetic analysis of AlAXEase and reported SGNH-type AcXEs. The tree was built by the neighbor-joining method with a JTT matrix–based mode using 112 amino acid positions. Bootstrap analysis of 1000 replicates is executed, and values above 50% are shown. AcXEs with structures are indicated by black circles. AcXEs, acetyl xylan esterases; AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.
      Multiple sequence alignment showed that AlAXEase contains the four characteristic sequence blocks of SGNH hydrolases, blocks I, II, III, and V (Fig. 2), further supporting that AlAXEase is a SGNH hydrolase. AlAXEase has a catalytic triad possibly formed by Ser32, Asp200, and His203 (Fig. 2). The catalytic Ser32 is located in the conserved GDSxT motif (block I) close to the N terminus, while Asp200 and His203 are located in the conserved DxxHL(P) motif (block V) (Fig. 2). Residues Gly69 and Asn98 were predicted to be involved in the oxyanion hole, which are located in blocks II and III, respectively (Fig. 2).
      Figure thumbnail gr2
      Figure 2Multiple sequence alignment of AlAXEase and reported SGNH-type AcXEs with structures. Using ESPript, secondary structures of AlAXEase are shown above alignment and secondary structures of Axe2 (PDB code 3W7V) under alignment. Helices are indicated by squiggles, β strands by arrows, turns by TT letters, and 310-helices by η letters. Identical amino acid residues are shown in white on a black shadow, and similar residues are in bold black. Stars represent residues belonging to the catalytic triad, and circles represent oxyanion hole residues. The four conserved sequence blocks in SGNH hydrolases are boxed by red dashed lines. The catalytic loop in AlAXEase and the corresponding loops in other SGNH-type AcXEs are boxed by green solid lines. AcXEs, acetyl xylan esterases; AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T; PDB, Protein Data Bank.

      AlAXEase is a cold-adapted acetyl xylan esterase with unusual thermostability

      AlAXEase without the predicted signal peptide was overexpressed in Escherichia coli BL21 (DE3) with the coexpression of the chaperone protein groES-groEL and purified. The recombinant AlAXEase with a calculated molecular mass of 23.5 kDa could hydrolyze p-nitrophenyl acetate (pNPC2), 1-napthyl acetate, and phenyl acetate but showed no detectable activity against pNP-acylesters with an acyl chain length of more than two carbon atoms (Table 1), suggesting that AlAXEase may have a small substrate-binding pocket. Using pNPC2 as the substrate, AlAXEase exhibited the highest activity at 30 °C and retained more than 70% of the highest activity at 0 °C (Fig. 3A), indicating that it is a cold-adapted enzyme. However, AlAXEase displayed unexpected tolerance to heat treatment, retaining 75% of the highest activity at 50 °C and 45% at 60 °C after 1 h incubation (Fig. 3B). Moreover, AlAXEase had a relatively high Tm value of 56 °C (Fig. 3C). These results indicate that the cold-adapted AlAXEase has unusual thermal stability. The cold-adapted AlAXEase was also resistant to mechanic stirring (Fig. S1). AlAXEase exhibited the highest activity at pH 9.0 and was stable in a range of pH 5.0 to 11.0 (Fig. 3D and Fig. S2). AlAXEase is also a halotolerant enzyme, whose activity was not influenced by 3.0 M NaCl (Fig. 3E). Among all the tested metal ions, only 10 mM of Cu2+, Fe2+, or Fe3+ severely inhibited AlAXEase activity, whereas the other metal ions had no or weak inhibitory effect on AlAXEase activity (Table 2). AlAXEase activity was not influenced by the metal chelator EDTA but severely inhibited by 10 mM PMSF (Table 2), suggesting that AlAXEase is a serine hydrolase.
      Table 1The substrate specificity of AlAXEase
      SubstrateSpecific activity (U/mg)
      p-Nitrophenyl acetate9.10 ± 0.09
      p-Nitrophenyl butyrate--
      Undetectable.
      p-Nitrophenyl caproate--
      Undetectable.
      p-Nitrophenyl caprylate--
      Undetectable.
      1-Napthyl acetate2.21 ± 0.18
      Phenyl acetate2.23 ± 0.44
      Isopropenyl acetate--
      Undetectable.
      Menthyl acetate0.40 ± 0.09
      Florfenicol0.07 ± 0.01
      Ethyl 2-chlorobenzoate0.10 ± 0.01
      Ethyl 4-chloro-3-hydroxybutanoate0.10 ± 0.01
      β-D-galactose pentaacetate3.63 ± 0.01
      β-D-glucose pentaacetate3.89 ± 0.06
      Sucrose octaacetate3.61 ± 0.34
      1,2,3,5-Tetra-O-acetyl-D-xylofuranose3.02 ± 0.21
      1,2,3,4-Tetra-O-acetyl-D-xylopyranose3.88 ± 0.14
      Benzyl β-D-xylobioside pentaacetate0.38 ± 0.03
      Xylan (partially acetylated)0.29 ± 0.03
      N-acetyl-D-glucosamine--
      Undetectable.
      a Undetectable.
      Figure thumbnail gr3
      Figure 3Biochemical characterization of AlAXEase. A, the effect of the temperature on the activity (solid line) and stability (dashed line) of AlAXEase. B, the effect of the temperature on the stability of AlAXEase. The enzyme was incubated at 40 °C, 50 °C, and 60 °C for different time intervals, and the residual activity was measured at pH 8.0 and 30 °C. C, thermal unfolding of AlAXEase and its mutant monitored by CD. The CD was monitored at 222 nm. The temperature was monitored using an internal sensor with a gradient of 1.0 °C per min. The inset shows the first derivative of the CD signal versus temperature. The data shown are representative of results of triplicate experiments. D, the effect of pH on the activity (solid line) and stability (dashed line) of AlAXEase. For stability, the enzyme was incubated in buffers ranging from pH 2.0 to 12.0 at 0 °C for 1 h, and the residual activity was measured at pH 8.0 and 30 °C. E, the effect of NaCl on the activity (solid line) and stability (dashed line) of AlAXEase. For stability, the enzyme was incubated at 0 °C for 1 h in buffers containing NaCl ranging from 0 to 4.8 M, and the residual activity was measured at pH 8.0 and 30 °C. F, kinetic parameters of AlAXEase against different acetylated monosaccharides. Enzyme kinetic assays of AlAXEase were carried out at pH 9.0 (20 mM Hepes) using 1,2,3,4-tetra-O-acetyl-D-xylopyranose, 1,2,3,5-tetra-O-acetyl-D-xylofuranose, β-D-glucose pentaacetate, and β-D-galactose pentaacetate at concentrations from 0.5 to 20 mM, respectively. The Km and kcat/Km values of AlAXEase against 1,2,3,4-tetra-O-acetyl-D-xylopyranose are considered to be 100%. In panels A, B, D, E, and F, the graphs show data from triplicate experiments (mean ± SD). AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.
      Table 2Effects of metal ions and potential inhibitors on AlAXEase activity
      CompoundRelative/residual activity (%)
      1 mM10 mM
      K+104.1 ± 0.7111.4 ± 1.85
      Li+105.9 ± 0.961.0 ± 2.9
      Ba2+112.1 ± 2.4103.1 ± 2.2
      Ca2+110.1 ± 1.5136.0 ± 1.9
      Co2+102.1 ± 0.770.8 ± 1.7
      Cu2+84.7 ± 0.34.85 ± 0.3
      Fe2+125.3 ± 3.45.3 ± 4.5
      Mg2+110.0 ± 2.0118.2 ± 1.1
      Mn2+107.1 ± 0.9127.1 ± 1.4
      Ni2+105.9 ± 0.961.0 ± 2.9
      Sr2+108.4 ± 1.1111.5 ± 1.7
      Zn2+105.1 ± 1.259.3 ± 0.5
      Fe3+111.4 ± 6.0--
      Undetectable.
      EDTA104.4 ± 1.7101.0 ± 1.5
      PMSF77.5 ± 1.138.4 ± 1.1
      a Undetectable.
      To reveal the natural substrates of AlAXEase, we also measured the activity of AlAXEase against different kinds of acetylated carbohydrates (Table 1). Similar to the acetyl xylan esterase Axe2 from G. stearothermophilus (Table S1), AlAXEase could deacetylate many acetylated monosaccharides and disaccharides including galactose, glucose, xylose in furanose and pyranose configurations, sucrose, and xylobioside, as well as partially acetylated xylan, with the highest activity toward acetylated glucose and xylopyranose (Table 1), indicating that AlAXEase is a CE. AlAXEase hardly degraded N-acetyl-D-glucosamine (Table 1), suggesting its high specificity for the O-acetyl groups rather than the N-acetyl groups of acetylated carbohydrates. Further kinetic analysis revealed that, among the acetylated monosaccharides, acetylated xylopyranose is the optimal substrate of AlAXEase, to which AlAXEase showed the highest substrate affinity and the highest catalytic efficiency (kcat/Km) (Fig. 3F and Table S2). Moreover, AlAXEase could hydrolyze both acetylated xylobioside and acetyl xylan (Table 1). All these data indicate that AlAXEase is an acetyl xylan esterase.

      Analysis of the overall structure and the active site of AlAXEase

      To reveal the underlying cold adaption mechanism of AlAXEase, we solved the crystal structure of WT AlAXEase by the molecular replacement method using selenomethionine (SeMet)-AlAXEase structure as the starting model because of the low sequence identities (lower than 24%) shared by AlAXEase and proteins with available structures in the Protein Data Bank (PDB) database. The crystal of AlAXEase belongs to the P1211 space group, and the structure of AlAXEase was solved at 2.50 Å resolution. The statistics for refinement are summarized in Table 3. Structural data show that each asymmetric unit contains four AlAXEase molecules (Fig. 4A). Gel filtration analysis showed that AlAXEase tends to form large oligomers in solutions (Fig. 4B), and dynamic light scattering (DLS) analysis indicated that AlAXEase forms stable tetramers in solutions (Fig. 4C).
      Table 3Data collection and refinement statistics of WT AlAXEase and SeMet-AlAXEase
      ParametersAlAXEaseSeMet-AlAXEase
      Data collection
       Space groupP1211P1211
       Unit cell dimensions
      a, b, c (Å)76.87, 80.61, 82.0672.22, 79.04, 81.80
      α, β, γ (°)90, 103.132, 9090, 104.28, 90
       Wavelength (Å)0.97910.9791
       Resolution range (Å)50.00–2.50 (2.54–2.50)
      Numbers in parentheses refer to data in the highest resolution shell.
      50.00–2.30 (2.34–2.30)
       Redundancy3.4 (3.5)3.0 (2.3)
       Completeness (%)98.891.6
      Rmerge
      Rmerge = ΣhklΣi|I(hkl)i - <I(hkl)>|/ΣhklΣi < I(hkl)i>.
      0.137 (0.306)0.153 (0.437)
       I/sigma8.25 (2.67)8.94 (1.27)
      Refinement statistics
       Resolution range (Å)42.07–2.51 (2.60–2.51)
       Rwork (%)17.92 (20.02)
       Rfree (%)23.54 (26.95)
       B-factor (Å2)
       Macromolecules28.09
       Solvent29.64
       RMSD from ideal geometry
      Bond lengths (Å)0.01
      Bond angles (°)1.02
       Ramachandran plot (%)
      Favored (%)93.91
      Allowed (%)6.09
      a Numbers in parentheses refer to data in the highest resolution shell.
      b Rmerge = ΣhklΣi|I(hkl)i - <I(hkl)>|/ΣhklΣi < I(hkl)i>.
      Figure thumbnail gr4
      Figure 4Overall structural analysis of AlAXEase. A, overall structure of tetrameric AlAXEase in one asymmetric unit. B, gel filtration analysis of AlAXEase. Aldolase (158 kDa), protein E40 (137 kDa) (
      • Li P.Y.
      • Chen X.L.
      • Ji P.
      • Li C.Y.
      • Wang P.
      • Zhang Y.
      • Xie B.B.
      • Qin Q.L.
      • Su H.N.
      • Zhou B.C.
      • Zhang Y.Z.
      • Zhang X.Y.
      Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.
      ), and protein DddP (110 kDa) (
      • Wang P.
      • Chen X.L.
      • Li C.Y.
      • Gao X.
      • Zhu D.Y.
      • Xie B.B.
      • Qin Q.L.
      • Zhang X.Y.
      • Su H.N.
      • Zhou B.C.
      • Xun L.Y.
      • Zhang Y.Z.
      Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial dimethylsulfoniopropionate lyase: A new enzyme from an old fold.
      ) were used as protein size markers. The theoretical molecular weight of monomeric AlAXEase without signal peptide is 23.5 kDa. C, DLS analysis of AlAXEase. D, superimposition of AlAXEase and other SGNH-type enzymes. AlAXEase is colored in green, the uncharacterized GDSL protein (PDB code 3RJT) from Alicyclobacillus acidocaldarius in cyan, CtCes3 (PDB code 2VPT) in magenta, and Axe2 (PDB code 3W7V) in yellow. E, overall structure of monomeric AlAXEase. The monomer has four β-sheets and eight α-helices. The catalytic triad residues (Ser32, Asp200, and His203) and the oxyanion hole residues (Gly69 and Asn98) are shown as sticks. The catalytic loop is colored in blue. F, surface view of monomeric AlAXEase. Active site residues Ser32, Gly69, Asn98, and His203 are colored in red, yellow, green, and magenta, respectively, and the catalytic loop in blue. AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T; DLS, dynamic light scattering.
      The overall structure of AlAXEase monomer is similar to those of other SGNH-type AcXEs (Fig. 4D), most closely resembling the structures of an uncharacterized GDSL protein (PDB code 3RJT) from Alicyclobacillus acidocaldarius and Axe2 (PDB code 3W7V) from G. stearothermophilus (
      • Lansky S.
      • Alalouf O.
      • Solomon H.V.
      • Alhassid A.
      • Govada L.
      • Chayen N.E.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
      ), with the RMSD of 1.34 Å (150 monomer Cα atoms) and 2.63 Å (147 monomer Cα atoms), respectively. Monomeric AlAXEase shows a typical SGNH hydrolase fold, consisting of a central four-stranded parallel sheet flanked by two layers of helices (Fig. 4E). Similar to most AcXEs (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ,
      • Correia M.A.S.
      • Prates J.A.M.
      • Bras J.
      • Fontes C.M.G.A.
      • Newman J.A.
      • Lewis R.J.
      • Gilbert H.J.
      • Flint J.E.
      Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition.
      ,
      • Lansky S.
      • Alalouf O.
      • Solomon H.V.
      • Alhassid A.
      • Govada L.
      • Chayen N.E.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
      ), AlAXEase has a catalytic triad formed by residues Ser32, Asp200, and His203, which are all located on the protein surface (Fig. 4F). Ser32 is situated on the terminus of α1, while Asp200 and His203 are located in a long surface loop between α7 and α8 (Fig. 4E). Mutation of these residues to Ala led to extremely low or no enzymatic activity (Table 4), demonstrating their key roles in the catalysis. The oxyanion hole is composed of two solvent-exposed residues, Gly69 and Asn98 (Fig. 4F). Both mutations G69A and N98A had a small impact on the Km, but significantly decreased the kcat of AlAXEase (Table 4), consistent with that the oxyanion hole residues are involved in stabilizing the tetrahedral intermediates in the reaction process through their main-chain nitrogen atoms (
      • Pfeffer J.M.
      • Weadge J.T.
      • Clarke A.J.
      Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.
      ). The catalytic residues and the oxyanion hole residues together with their adjacent residues form a shallow substrate-binding pocket of AlAXEase (Fig. 4F).
      Table 4Kinetic parameters of AlAXEase and its mutants against 1,2,3,4-tetra-O-acetyl-D-xylopyranose
      EnzymeTemperature (°C)Vmax (μM/min/mg)Km (mM)kcat (s−1)kcat/Km (mM−1 s−1)
      WT105.2 ± 0.173.4 ± 0.082.0 ± 0.070.60 (83%)
      WT207.2 ± 0.014.5 ± 0.352.8 ± 0.010.63 (88%)
      WT309.2 ± 0.465.0 ± 0.333.6 ± 0.180.72 (100%)
      WT404.6 ± 0.675.6 ± 0.571.8 ± 0.260.33 (46%)
      WT501.0 ± 0.066.1 ± 0.160.41 ± 0.020.07 (10%)
      S32A30--
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      G69A303.1 ± 0.045.3 ± 0.301.2 ± 0.010.23 (31%)
      N98A300.05 ± 0.016.0 ± 0.360.02 ± 0.010.01 (1.4%)
      D200A300.05 ± 0.025.0 ± 0.160.02 ± 0.010.01 (1.4%)
      H203A30--
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      E190A104.4 ± 0.373.6 ± 0.251.7 ± 0.140.45 (63%)
      E190A205.7 ± 0.955.6 ± 0.212.2 ± 0.370.49 (68%)
      E190A302.0 ± 0.066.0 ± 0.160.77 ± 0.020.13 (18%)
      E190A400.48 ± 0.106.7 ± 0.130.19 ± 0.040.03 (4.2%)
      E190A50--
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      a Undetectable.
      In the AlAXEase tetramer, the interface between chains B and C is the largest, followed by the interface between chains C and D, and the remaining interfaces involving chain A are the least (Fig. 5A). The dimerization interface between chains B and C is mainly stabilized by hydrogen bonds and salt bridges involving eight residues Lys (71, 114), Gly (69, 107, 109), Asp (74, 111), and Thr108 from the interactive monomers (Fig. 5B), and the interface between chains C and D mainly by four hydrophilic residues Asp146, His147, Asn156, and Asn160 (Fig. 5C).
      Figure thumbnail gr5
      Figure 5Oligomerization of AlAXEase. A, surface view of tetrameric AlAXEase. The four chains of AlAXEase are shown in different colors, and the catalytic triad and the residue Glu190 in the catalytic loop of each chain are highlighted in red and blue, respectively. B, the hydrogen-bond network between chains B and C. Residues in chain B are shown in cyan, and residues in chain C in magenta. For both chains, catalytic triad residues are shown in ball-and-stick representation. C, the hydrogen-bond network between chains C and D. Residues in chain C are shown in magenta and residues in chain D in yellow. For both chains, catalytic triad residues are shown in ball-and-stick representation. AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.

      Structural basis for the high thermostability of AlAXEase

      Among all the characterized proteins, the sequence and topological structure of AlAXEase are most closely related to those of Axe2 albeit with a low similarity of 24% (Figs. 1 and 4). Axe2 is a mesophilic enzyme with the highest activity between 50 °C and 60 °C and a Tm value of 72 °C (
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ). The most common determinants for increased thermostability of hyperthermophilic proteins are more noncovalent stabilizing interactions (
      • Siddiqui K.S.
      • Cavicchioli R.
      Cold-adapted enzymes.
      ,
      • Tang M.A.
      • Motoshima H.
      • Watanabe K.
      Fluorescence studies on the stability, flexibility and substrate-induced conformational changes of acetate kinases from psychrophilic and mesophilic bacteria.
      ,
      • Struvay C.
      • Feller G.
      Optimization to low temperature activity in psychrophilic enzymes.
      ). At the monomer level, AlAXEase has similar numbers of hydrogen bonds and ionic interactions as Axe2 (Table 5), suggesting that AlAXEase has a high overall stability, thus leading to the high thermostability of AlAXEase. From psychrophiles to mesophiles to thermophiles, a clear trend can be observed that shows an increase in the number of ionic attractions on the protein surface (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ,
      • Sanchez-Ruiz J.M.
      • Makhatadze G.I.
      To charge or not to charge?.
      ). Compared with Axe2, AlAXEase has a more positively charged interface near its active site (Fig. 6A), fewer stabilizing prolines, and more thermally labile residues asparagine and lysine on its surface (Table 5 and Fig. 6, B and C), which may result in the lower thermostability of AlAXEase than Axe2.
      Table 5Structure and sequence comparison of AlAXEase and Axe2
      Sequence/structural informationAlAXEase
      No. of hydrogen bonds and ion pairs were calculated based on the four chains in the crystal structure of WT AlAXEase.
      Axe2
      No. of hydrogen bonds and ion pairs were calculated based on the two chains in the crystal structure of Axe2.
      Tm (°C)5672
      Topt (°C)3050–60
      Recombinant protein sequence length208219
      Hydrophobic residues
      Hydrophobic residues A, V, L, I, W, F, P, and M.
      (%)
      38.545.7
      Polar residues
      Polar residues G, S, T, Y, N, Q, and C.
      (%)
      31.727.9
      Charged residues
      Charged residues R, K, H, D, and E.
      (%)
      29.826.5
      Net charges (Arg + Lys - Asp - Glu)−3−3
      No. of Gly/Met/Pro16/5/419/7/9
      No. of Asn/Gln16/86/7
      Arg/(Arg + Lys)0.2960.577
      Sequence identity to AlAXEase100%24%
      PDB entryThis study3W7V
      Resolution (Å)2.501.85
      No. of residues per monomer in the crystal structure199 ± 1219 ± 0
      RMSD (Å) (no. of residues)2.69 ± 0.09 (148 ± 2)
      No. of hydrogen bonds per residue in monomer2.374 ± 0.042.297 ± 0.04
      No. of side-chain to side-chain hydrogen bonds per residue0.398 ± 0.040.416 ± 0.05
      No. of side-chain to main-chain hydrogen bonds per residue0.550 ± 0.020.506 ± 0.03
      No. of main-chain to main-chain hydrogen bonds per residue1.426 ± 0.021.374 ± 0.03
      No. of ion pairs per monomer at 4 Å12 ± 1.614 ± 2.8
      No. of ion pairs per monomer at 6 Å22.5 ± 0.626 ± 2.8
      No. of ion pairs per residue (4 Å)0.060 ± 0.010.064 ± 0.01
      a No. of hydrogen bonds and ion pairs were calculated based on the four chains in the crystal structure of WT AlAXEase.
      b No. of hydrogen bonds and ion pairs were calculated based on the two chains in the crystal structure of Axe2.
      c Hydrophobic residues A, V, L, I, W, F, P, and M.
      d Polar residues G, S, T, Y, N, Q, and C.
      e Charged residues R, K, H, D, and E.
      Figure thumbnail gr6
      Figure 6Comparative structural analysis of cold-adapted AlAXEase and mesophilic Axe2. A, electrostatic surfaces of AlAXEase and Axe2. The positively charged regions are shown in blue and the negatively charged regions in red. The catalytic cavities of AlAXEase and Axe2 are marked with yellow circles. B, cartoon view of AlAXEase and Axe2. For both AlAXEase and Axe2, stabilizing residues Arg and Pro are shown as red lines, destabilizing residues Asn and Lys as blue sticks, and catalytic triad residues in ball-and-stick representation. Catalytic loop in AlAXEase is colored in magenta, and the counterpart in Axe2 in yellow. C, B factor analysis of AlAXEase and Axe2. The thicker coils show higher flexibility than other parts of the protein. The catalytic triad residues of AlAXEase and Axe2 are shown as sticks. AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.
      In addition, oligomerization also contributes to the thermal stability of proteins (
      • Tanaka Y.
      • Tsumoto K.
      • Yasutake Y.
      • Umetsu M.
      • Yao M.
      • Fukada H.
      • Tanaka I.
      • Kumagai I.
      How oligomerization contributes to the thermostability of an Archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii.
      ,
      • Ogasahara K.
      • Ishida M.
      • Yutani K.
      Stimulated interaction between alpha and beta subunits of tryptophan synthase from hyperthermophile enhances its thermal stability.
      ). Axe2 forms a ‘doughnut-shaped’ homo-octamer with two staggered tetrameric rings both in the crystal and in solution, and the oligomerization of Axe2 is mainly stabilized by a cluster of hydrogen bonds and π-stacking interactions involving residues near the active sites of all eight monomers (
      • Lansky S.
      • Alalouf O.
      • Solomon H.V.
      • Alhassid A.
      • Govada L.
      • Chayen N.E.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
      ). Similar to Axe2, AlAXEase also forms large oligomers. AlAXEase forms tetramers both in the crystal and solution (Fig. 4), which may play a role in maintaining the structural stability and thermostability of AlAXEase. Different from Axe2 octamers, AlAXEase tetramers are mainly maintained by residues far away from their active sites (Fig. 5). Moreover, the smaller oligomerization interfaces of AlAXEase than those of Axe2 suggest that AlAXEase tetramers are less compact than Axe2 octamers, which may also contribute to the lower thermostability of AlAXEase.

      AlAXEase has a long and flexible catalytic loop around its active site

      At the monomer level, the largest structural difference between AlAXEase and Axe2 is that the loop containing the catalytic residues Asp200 and His203 (18 residues in length) in AlAXEase is much longer than the corresponding one in Axe2 (8 residues in length) (Fig. 7A). The catalytic loop of AlAXEase is also the longest one among all the characterized AcXEs with solved structures (Fig. 2). Based on the B factor analysis, the flexible regions in AlAXEase and Axe2 are similar, except that the active site of AlAXEase is more flexible, especially the long catalytic loop (Fig. 6C). In AlAXEase, the catalytic loop is mainly stabilized by forming hydrogen bonds with two residues (Glu143 and Asp146) in the loop between β4 and α6 and hydrophobic interactions involving four residues (Ile196, Leu197, Val202, and Leu204) in the catalytic loop and eight hydrophobic residues in the other regions of AlAXEase (Fig. 7, B and D). For Axe2, similar hydrogen bonds and hydrophobic interactions are found to stabilize its short catalytic loop (Fig. 7, C and E). However, AlAXEase has less hydrophobic interactions (a 12-member cluster) around the catalytic loop than Axe2 (a 16-member cluster) (Fig. 7F). Moreover, no interaction is present to maintain the structure of the region 192KDRG195 in the catalytic loop of AlAXEase (Fig. 7, B and D), and this region and its upstream residues are rich in destabilizing asparagine and lysine residues (Fig. 6B). In addition, the catalytic loop in Axe2 also forms intermolecular hydrogen bonds between interactive monomers (
      • Lansky S.
      • Alalouf O.
      • Solomon H.V.
      • Alhassid A.
      • Govada L.
      • Chayen N.E.
      • Belrhali H.
      • Shoham Y.
      • Shoham G.
      A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
      ), which, however, are absent from AlAXEase. All these differences make the catalytic loop of AlAXEase more flexible than that in Axe2, which would improve the flexibility of the catalytic center and lead to the high activity of AlAXEase at low temperatures. When the catalytic loop in AlAXEase was shortened (mutants △2 and △3 in Fig. 8A) or substituted by the short catalytic loop of Axe2 (mutants L1 and L2 in Fig. 8A), all the mutants were inactive (Fig. 8A), indicating that the length and flexibility of the catalytic loop is important for maintaining the catalytic activity of AlAXEase.
      Figure thumbnail gr7
      Figure 7Analysis of the interactions between the catalytic loop and other regions in AlAXEase and Axe2. A, the superimposition of AlAXEase (green) and Axe2 (cyan). Catalytic loop in AlAXEase is colored in magenta, and the counterpart in Axe2 in yellow. For both AlAXEase and Axe2, the catalytic triad residues and the oxyanion hole residues are shown as sticks. B, the hydrogen-bond network between the catalytic loop (magenta) and other regions (green) in AlAXEase. Key residues involved in these interactions are shown as sticks. C, the hydrogen-bond network between the catalytic loop (yellow) and other regions (cyan) in Axe2. Key residues involved in these interactions are shown as sticks. D, the hydrophobic interactions between the catalytic loop (magenta) and other regions (green) in AlAXEase. Key hydrophobic residues are shown as sticks. E, the hydrophobic interactions between the catalytic loop (yellow) and other regions (cyan) in Axe2. Key hydrophobic residues are shown as sticks. In panels BE, the catalytic triads are in ball-and-stick representation. F, the superimposition of hydrophobic residues in AlAXEase and Axe2 involved in the hydrophobic interactions between the catalytic loop (magenta for AlAXEase and yellow for Axe2) and other regions (green for AlAXEase and cyan for Axe2). AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.
      Figure thumbnail gr8
      Figure 8Analyses of the thermodependence of activity and thermostability of mutant E190A. A, enzymatic activities of the mutants of AlAXEase. Mutant L1 with mutation to replace residues 185LKNNPENKDRGILTR199 in the catalytic loop of AlAXEase with 181KTLYPAALAW190 of Axe2, mutant L2 with mutation to replace residues 185LKNNPENKDRGI196 of AlAXEase with 181KTLYPAA187 of Axe2, mutant Δ2 with mutation to delete residues Asn188 and Pro189 of AlAXEase, and mutant Δ3 with mutation to delete residues Asn188, Pro189, and Glu190 of AlAXEase. The activities of WT AlAXEase and mutant E190A were measured under their respective optimum temperatures. For all other mutants, no enzymatic activity was detected at temperatures ranging from 0 to 60 °C. B, the effect of temperature on the activity (solid line) and stability (dashed line) of mutant E190A. For stability, the enzyme was incubated from 0 to 60 °C for 1 h, and the residual activity was measured under optimal conditions. C, the effect of the temperature on the stability of mutant E190A. The enzyme was incubated at 20 °C and 30 °C for different time intervals, and the residual activity was measured under optimal conditions. In panels AC, the graphs show data from triplicate experiments (mean ± SD). AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.

      The catalytic loop contributes to the cold-adapted characteristics of AlAXEase by modulating the distance between the catalytic residues Ser32 and His203

      To further investigate the role of the catalytic loop in the cold adaptation of AlAXEase, site-directed mutagenesis on the residue Glu190 in the catalytic loop with the highest B factor was performed. Compared with WT AlAXEase, mutant E190A had a lower optimum temperature (Topt) of 20 °C (Figs. 3A and 8B). At 10 °C, mutant E190A retained 92% of its maximal catalytic efficiency (kcat/Km), higher than that of the WT (83%) (Table 4), suggesting that mutant E190A is more active than the WT at low temperatures. Mutant E190A also had a lower thermostability, quite unstable at temperatures above 20 °C (Fig. 8, B and C). These data suggest that mutant E190A is more cold-adapted than WT AlAXEase.
      Then, structural analyses and MD simulations of WT AlAXEase and its mutant E190A at different temperatures were carried out to further probe the molecular mechanism for the cold adaptation of AlAXEase (Figs. 9 and 10 and Fig. S3). At all simulated temperatures, no significant differences were observed in the RMSD values of the backbone atoms of both enzymes (Fig. 10A), suggesting that the introduction of the E190A mutation in the catalytic loop has little impact on the overall structures of AlAXEase monomers under different temperatures. However, the fluorescence peak position of AlAXEase began to change with a blue shift at 60 °C, and that of mutant E190A at 20 °C (Fig. 9A), indicating that the tertiary structure of the mutant is less rigid and less stable than that of the WT against high temperatures. Moreover, different from the WT (with a Tm value of 56 °C), mutant E190A presented two thermal transitions (Fig. 3C), one at ∼30 °C and the other at ∼60 °C, suggesting that some regions of the enzyme unfold first at a low temperature, followed by the unfolding of the remaining regions at relatively high temperature. These data suggest that the introduced mutation E190A may cause an increased flexibility in local rather than overall structure of AlAXEase to enhance its cold adaptation.
      Figure thumbnail gr9
      Figure 9Effect of different temperatures on the structures of WT AlAXEase and its mutant E190A. A, fluorescence spectra of AlAXEase and its mutant E190A after incubation at different temperatures for 1 h. B, gel filtration analysis of AlAXEase and its mutant E190A after incubation at different temperatures for 30 min. Aldolase (158 kDa), conalbumin (75 kDa), and carbonic anhydrase (29 kDa) were used as protein size markers. The WT AlAXEase kept tetramers at all the temperatures measured, whereas a part of the tetramers of mutant E190A were depolymerized to monomers at its optimum temperature of 20 °C. AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T.
      Figure thumbnail gr10
      Figure 10MD simulations of AlAXEase and its mutant E190A at different temperatures. A, the backbone RMSD values of the MD simulations for AlAXEase and its mutant E190A. RMSD values at 280 K and 400 K and that from 400 K back to 280 K are shown in black, red, and blue, respectively. B, the residue RMSF values of the MD simulations for AlAXEase and its mutant E190A. RMSF values at 280 K and 400 K and that from 400 K back to 280 K are shown in black, red, and blue, respectively. AlAXEase, a cold-adapted AcXE from Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T; RMSF, root mean square fluctuation.
      Root mean square fluctuation values often reflect the fluctuation of individual residues during the MD simulation process (
      • Martinez L.
      Automatic identification of mobile and rigid substructures in molecular dynamics simulations and fractional structural fluctuation analysis.
      ). As shown in Figure 10B, both AlAXEase and its mutant contain three major unstable regions, including (1) the loop between β2 and α3 and the initial proportion of α3 (residues 67–76), (2) the latter part of α4 and the loop between α4 and α5 (residues 103–111), and (3) the region near the active site involving the catalytic loop. The latter part of the loop between β4 and α6 (residues 143–152) is also unstable in AlAXEase but stable in the mutant. Except for the unstable regions near the active site, all other unstable regions are located in the oligomerization interfaces of AlAXEase (Fig. 5), suggesting that heat treatment may influence the oligomerization of protein. Notably, at 45 °C, AlAXEase lost most of the enzymatic activity (Fig. 3A) but still retained tetramers (Fig. 9B), demonstrating that the cold-adapted characteristics of AlAXEase come from the flexibility of its monomeric rather than oligomeric structure. Different from the WT, a part of the tetramers of mutant E190A were depolymerized to monomers at its Topt of 20 °C (Fig. 9B), suggesting that the introduced mutation E190A makes AlAXEase tetramers tend to depolymerize to decrease its thermostability.
      MD simulations also showed that the regions around the active sites of both AlAXEase and mutant E190A become flexible at a high temperature (Fig. 10B). Compared with the small unstable part of the catalytic loop (residues 201 and 202) in AlAXEase, mutant E190A possessed a larger unstable region around the active site including the α7 and the following long catalytic loop (residues 180–194, 201, and 202) (Fig. 10B). It has been found that high flexibility, particularly around the active site, is usually associated with low substrate affinity in cold-adapted enzymes (
      • Siddiqui K.S.
      • Cavicchioli R.
      Cold-adapted enzymes.
      ,
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ). Similarly, compared with Axe2, AlAXEase and mutant E190A showed increased Km values, and the Km values of mutant E190A were higher than those of AlAXEase (Table 4 and Table S3), further indicating a flexible active site in AlAXEase and a more flexible active site in the mutant. Different from the reversible active site of AlAXEase, the active site of mutant E190A was irreversibly disrupted at a high temperature and thus led to the distortion of the α7 helix upstream of the catalytic loop to a random coil structure (Fig. 10B), indicating that the introduced mutation E190A in the catalytic loop makes the region around the active site more susceptible to thermal denaturation than other regions of AlAXEase as shown by CD (Fig. 3C).
      During the MD simulation, we measured the distances between the key residues in the active site to further assess the effect of heat treatment on the active site of AlAXEase. At all simulated temperatures, the distance variations between the key residues in the active site were kept at a very small range in AlAXEase and mutant E190A except for the distance between the two catalytic residues, Ser32 and His203 (Table 6). For both enzymes, the distances between Ser32 and His203 at 400 K were significantly enlarged compared with those at 280 K, and these distance variations were irreversible when the proteins were cooled from 400 K to 280 K (Table 6). The enlargement of the distance between Ser32 and His203 resulted in the reduction in both activity and substrate affinity of both enzymes at temperatures higher than their respective Topt as indicated in Table 4. Notably, at 280 K, the distance between Ser32 and His203 in mutant E190A (6.5 ± 1.0 Å) is greater than that in the WT (4.3 ± 0.4 Å) (Table 6). Moreover, mutant E190A lost its catalytic activity and substrate-binding ability at a temperature (40 °C) lower than that for the WT (50 °C) (Table 4). All these results suggest that the flexible catalytic loop contributes to the cold-adapted characteristics (high catalytic activity and high substrate affinity at low temperatures) of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32, and that the introduced mutation E190A causes a further increase of flexibility in the catalytic loop of AlAXEase, leading to an improvement of its cold adaptation.
      Table 6The distances between key residues in the active sites of WT AlAXEase and its mutant E190A based on MD simulations
      EnzymeCrystal/MD simulationDistance (Å)
      The corresponding atom/group of a given residue used for distance calculation is shown in parentheses.
      S32 (Cα) - G69 (N)S32 (Cα) - N98 (N)S32 (OG) - H203 (NE2)D200 (OD1) - H203 (ND1)D200 (OD2) - H203 (ND1)
      WTCrystal
      The distances were calculated based on the active sites of four chains in the crystal structure of WT AlAXEase.
      5.0 ± 0.19.1 ± 0.13.6 ± 0.22.6 ± 0.33.5 ± 0.2
      280 K4.7 ± 0.28.6 ± 0.64.3 ± 0.44.3 ± 1.44.8 ± 1.7
      400 K5.7 ± 1.27.0 ± 0.510.5 ± 2.45.5 ± 2.25.5 ± 2.2
      400 K back to 280 K4.3 ± 0.36.8 ± 0.311.1 ± 1.44.3 ± 1.54.9 ± 1.3
      E190ACrystal--
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      --
      Undetectable.
      280 K6.2 ± 0.310.1 ± 0.36.5 ± 1.03.3 ± 0.32.9 ± 0.2
      400 K7.2 ± 1.57.4 ± 0.99.3 ± 2.14.4 ± 2.04.4 ± 2.0
      400 K back to 280 K7.7 ± 0.77.5 ± 0.48.2 ± 1.13.0 ± 0.53.3 ± 0.6
      Abbreviation: SeMet, selenomethionine.
      a The corresponding atom/group of a given residue used for distance calculation is shown in parentheses.
      b The distances were calculated based on the active sites of four chains in the crystal structure of WT AlAXEase.
      c Undetectable.

      Discussion

      AcXEs play important roles in both marine and terrestrial xylan degradation and recycling (
      • Adesioye F.A.
      • Makhalanyane T.P.
      • Biely P.
      • Cowan D.A.
      Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
      ). AcXEs, dominated by SGNH-type enzymes, are distributed in nine CE families in the CAZy database (
      • Lombard V.
      • Golaconda Ramulu H.
      • Drula E.
      • Coutinho P.M.
      • Henrissat B.
      The carbohydrate-active enzymes database (CAZy) in 2013.
      ) in addition to the recently discovered Axe2 family (
      • Alalouf O.
      • Balazs Y.
      • Volkinshtein M.
      • Grimpel Y.
      • Shoham G.
      • Shoham Y.
      A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
      ,
      • Soni S.
      • Sathe S.S.
      • Odaneth A.A.
      • Lali A.M.
      • Chandrayan S.K.
      SGNH hydrolase-type esterase domain containing Cbes-AcXE2: A novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.
      ). Compared with the extensive study on terrestrial mesophilic/thermophilic AcXEs, study on marine cold-adapted AcXEs is still scarce. Until now, owing to the lack of structural information, the cold adaption mechanisms for SGNH-type AcXEs are still unknown. In this study, a novel cold-adapted AcXE, AlAXEase, was characterized from the Arctic marine bacterium A. luteifluviistationis SM1504T. AlAXEase shares low sequence identities (≤24%) with characterized AcXEs, and phylogenetic analysis suggests that AlAXEase and its homologs represent a new SGNH-type CE family. AlAXEase had the highest activity at 30 °C and displayed high catalytic activity at 0 to 20 °C, showing its cold-adapted character. However, different from other cold-adapted enzymes that are generally thermolabile (
      • Zhao F.
      • Cao H.Y.
      • Zhao L.S.
      • Zhang Y.
      • Li C.Y.
      • Zhang Y.Z.
      • Li P.Y.
      • Wang P.
      • Chen X.L.
      A novel subfamily of endo-beta-1,4-glucanases in glycoside hydrolase family 10.
      ,
      • Rutkiewicz M.
      • Bujacz A.
      • Bujacz G.
      Structural features of cold-adapted dimeric GH2 beta-D-galactosidase from Arthrobacter sp. 32cB.
      ,
      • Lee C.W.
      • Kwon S.
      • Park S.H.
      • Kim B.Y.
      • Yoo W.
      • Ryu B.H.
      • Kim H.W.
      • Shin S.C.
      • Kim S.
      • Park H.
      • Kim T.D.
      • Lee J.H.
      Crystal structure and functional characterization of an esterase (EaEST) from Exiguobacterium antarcticum.
      ), AlAXEase has unusual thermostability, with a relatively high Tm value of 56 °C and stable at temperatures up to 50 °C, suggesting that the cold adaption strategy adopted by AlAXEase is different from other thermolabile cold-adapted enzymes.
      Most cold-adapted enzymes are highly flexible in their overall structures, leading to their high catalytic activity at low temperatures but low thermostability (
      • Siddiqui K.S.
      • Cavicchioli R.
      Cold-adapted enzymes.
      ). However, a few cold-adapted enzymes are also reported to be locally flexible without compromising the global stability of proteins (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ,
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ,
      • Le L.T.H.L.
      • Yoo W.
      • Jeon S.
      • Lee C.
      • Kim T.D.
      Biodiesel and flavor compound production using a novel promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from the psychrophilic bacterium Halocynthiibacter arcticus.
      ,
      • Lian K.
      • Leiros H.K.
      • Moe E.
      MutT from the fish pathogen Aliivibrio salmonicida is a cold-active nucleotide-pool sanitization enzyme with unexpectedly high thermostability.
      ,
      • Yang G.
      • De Santi C.
      • de Pascale D.
      • Pucciarelli S.
      • Pucciarelli S.
      • Miceli C.
      Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus.
      ). Biochemical and structural analyses suggested that AlAXEase has high overall stability but is flexible in the loop containing the catalytic residues Asp200 and His203 because of the reduced stabilizing hydrophobic interactions and increased destabilizing residues asparagine and lysine (Figs. 6 and 7). Further structural and enzyme kinetic analyses of WT AlAXEase and its mutant E190A combined with MD simulations at different temperatures revealed that, the flexible catalytic loop contributes to the cold-adapted characteristics of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. The cold-adapted enzymes CpPAH and DpIDH are also reported to be locally flexible around their active sites because of the disrupted hydrogen-bonding abilities for the cofactor BH4 (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ) and the increase in destabilizing residues such as methionine and charged amino acids (
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ), respectively. Both CpPAH and DpIDH have flexible active sites through increasing flexibilities in noncatalytic residues in their catalytic cavities, which contribute to their cold-adapted characteristics (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ,
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ). However, different from CpPAH, DpIDH, and other cold-adapted enzymes (
      • Leiros H.K.
      • Pey A.L.
      • Innselset M.
      • Moe E.
      • Leiros I.
      • Steen I.H.
      • Martinez A.
      Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
      ,
      • Fedoy A.E.
      • Yang N.
      • Martinez A.
      • Leiros H.K.
      • Steen I.H.
      Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
      ,
      • Kulakova L.
      • Galkin A.
      • Nakayama T.
      • Nishino T.
      • Esaki N.
      Cold-active esterase from Psychrobacter sp. Ant300: Gene cloning, characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability.
      ), the flexible active site of AlAXEase comes from the increased flexibilities in the catalytic residues Asp200 and His203 rather than noncatalytic residues. Therefore, the cold adaption mechanism of AlAXEase is different from those of other reported cold-adapted enzymes. The flexible active site contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic residues His203 and Ser32. These data indicate that optimization of the flexibility of the catalytic residues is also a strategy for cold adaptation of enzymes.
      The marine strain SM1504T where AlAXEase comes from was reported to be cold adapted, growing at temperatures between 4 °C and 30 °C (optimum of 20 °C) (
      • Li D.D.
      • Peng M.
      • Wang N.
      • Wang X.J.
      • Zhang X.Y.
      • Chen X.L.
      • Su H.N.
      • Zhang Y.Z.
      • Shi M.
      Arcticibacterium luteifluviistationis gen. nov., sp. nov., isolated from Arctic seawater.
      ). The cold adaptation of AlAXEase is consistent with the growth characteristics of strain SM1504T, suggesting that its structural and biochemical properties are optimized to low temperatures. Genomic analysis showed that this strain contains some genes encoding potential xylanases, arabinofuranosidases, and other xylan-degrading enzymes (
      • Li Y.
      • Guo X.H.
      • Dang Y.R.
      • Sun L.L.
      • Zhang X.Y.
      • Chen X.L.
      • Qin Q.L.
      • Wang P.
      Complete genome sequence of Arcticibacterium luteifluviistationis SM1504T, a cytophagaceae bacterium isolated from Arctic surface seawater.
      ). AlAXEase could hydrolyze many kinds of acetylated monosaccharides and disaccharides as well as xylan, with acetylated xylopyranose as the optimal substrate, suggesting that AlAXEase is likely involved in xylan/xylooligosaccharide degradation together with other xylan-degrading enzymes to provide carbon source and energy for its source strain. Moreover, the cold-adapted characteristics of AlAXEase with unusual thermostability may also help its source strain SM1504T adapt to the cold polar environment.

      Experimental procedures

      Gene cloning and mutagenesis

      Based on blasting analysis, a gene AlAXEase encoding a GDSL family lipolytic protein (GenBank Accession No. WP_111370902) was identified from the genome sequence of marine bacterium A. luteifluviistationis SM1504T. AlAXEase without the signal peptide sequence was amplified from the genomic DNA of strain SM1504T, and the amplified fragment was ligated into the vector pET22b. All of the site-directed mutations and the truncated mutations in AlAXEase were introduced with the QuikChange mutagenesis kit (
      • Liu H.
      • Naismith J.H.
      An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.
      ) using plasmid pET22b-AlAXEase as the template. All recombinant plasmids were verified by sequencing.

      Protein expression and purification

      WT AlAXEase protein and all mutants were expressed in E. coli BL21 (DE3) with the coexpression of the chaperone groES-groEL. The cells were cultured at 37 °C to an absorbance at 600 nm of 0.6 to 1.0 and then induced by the addition of 1 mM IPTG and 0.5 mg/ml L-arabinose at 20 °C for 16 h. Cells were collected and disrupted by a JN-02C French press (JNBIO) in 50 mM Tris HCl buffer (pH 8.0) containing 100 mM NaCl and 5 mM imidazole. After centrifugation at 15,000g for 1 h at 4 °C, the recombinant proteins were first purified by Ni affinity chromatography (Qiagen) and then by ion-exchange chromatography on a SOURCE 15Q column (GE healthcare). The eluted enzyme fractions were further purified by gel filtration chromatography on a Superdex 200 column (GE healthcare) with 10 mM Tris HCl buffer (pH 8.0) containing 100 mM NaCl. The target protein was collected, and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Scientific).

      Enzyme activity assay

      Esterase activity was measured as described (
      • Li P.Y.
      • Ji P.
      • Li C.Y.
      • Zhang Y.
      • Wang G.L.
      • Zhang X.Y.
      • Xie B.B.
      • Qin Q.L.
      • Chen X.L.
      • Zhou B.C.
      • Zhang Y.Z.
      Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family.
      ). The standard reaction system (1 ml) contained 50 mM Tris HCl buffer (pH 8.0), 0.02 ml of 10 mM pNP-acylesters (Sigma), and 0.02 ml enzyme with an appropriate concentration. After incubation at 30 °C for 5 min, the reaction was terminated by the addition of 0.1 ml 20% SDS (w/v). The absorbance of the reaction mixture at 405 nm was measured using a SpectraMax Plus384 microplate spectrophotometer (Molecular Devices). One unit of enzyme (U) is defined as the amount of enzyme required to liberate 1 μmol p-nitrophenol per minute.
      The CE activity of AlAXEase was determined by detecting the release of acetic acid using synthetic substrates 1-napthyl acetate, phenyl acetate, isopropenyl acetate, menthyl acetate, florfenicol, ethyl 2-chlorobenzoate, and ethyl 4-chloro-3-hydroxybutanoate as well as acetylated carbohydrates β-D-galactose pentaacetate, β-D-glucose pentaacetate, sucrose octaacetate, 1,2,3,5-tetra-O-acetyl-D-xylofuranose, 1,2,3,4-tetra-O-acetyl-D-xylopyranose, benzyl β-D-xylobioside pentaacetate, partially acetylated xylan, and N-acetyl-D-glucosamine. 1-Napthyl acetate, phenyl acetate, β-D-galactose pentaacetate, β-D-glucose pentaacetate, sucrose octaacetate, and N-acetyl-D-glucosamine were purchased from Sigma. Menthyl acetate, florfenicol, ethyl 2-chlorobenzoate, ethyl 4-chloro-3-hydroxybutanoate, and 1,2,3,4-tetra-O-acetyl-D-xylopyranose were purchased from Aladdin. 1,2,3,5-Tetra-O-acetyl-D-xylofuranose and benzyl β-D-xylobioside pentaacetate were purchased from Zzstandard, and the partially acetylated xylan from Megazyme. The standard assay system contained 0.01 ml of 20 mM substrate dissolved in 50 mM Tris-HCl buffer (pH 9.0) containing 40% (v/v) isopropyl alcohol, and 0.01 ml enzyme with appropriate concentration. The reaction took place for 1 h at 30 °C. The release of acetic acid was determined with an Acetic Acid (ACS Analyser Format) Assay Kit (Megazyme, Ireland) according to the manufacturer’s instructions. One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 μmol of acetic acid per minute.

      Biochemical characterization of AlAXEase and its mutants

      By using pNPC2 as the substrate, the biochemical characteristics of AlAXEase and its mutants were studied. The Topt for AlAXEase activity was measured in the temperature range of 0 to 60 °C at pH 8.0. For thermostability assay, the enzyme was incubated at 40 °C, 50 °C, and 60 °C for different periods, and then, the residual activity was measured at 30 °C. The optimum pH of AlAXEase was determined at 30 °C in the Britton–Robinson buffers ranging from pH 2.0 to pH 12.0. For pH stability assay, the enzyme was incubated in buffers with a pH range of 2.0 to 12.0 at 0 °C for 1 h, and then, the residual activity was measured at pH 8.0 and 30 °C. The effect of NaCl on AlAXEase activity was determined at NaCl concentrations ranging from 0 to 4.8 M. For salt tolerance assay, the enzyme was incubated at 0 °C for 1 h in buffers containing NaCl ranging from 0 to 4.8 M before the residual activity was measured at 30 °C. The effects of metal ions and potential inhibitors on AlAXEase activity were examined by the addition of various chemical agents to the reaction mixture.
      Enzyme kinetic assays of AlAXEase and its mutants were carried out at pH 9.0 (20 mM Hepes) using 1,2,3,4-tetra-O-acetyl-D-xylopyranose at concentrations from 0.5 to 20 mM. Kinetic parameters were calculated by nonlinear regression fit directly to the Michaelis–Menten equation using the Origin9.0 software.

      Crystallization, data collection, and structure determination

      Crystals suitable for X-ray diffraction were obtained using the hanging-drop vapor-diffusion method. WT AlAXEase crystals grew at 18 °C in the buffer containing 0.1 M succinic acid and 15% (w/v) PEG 3350 for 1 week. Selenomethionine-AlAXEase crystals grew at 18 °C in the buffer containing 0.2 M potassium thiocyanate, 0.1 M Bis-Tris propane (pH 7.5), and 20% (w/v) PEG 3350 for 1 week. X-ray diffraction data were collected on the BL17U1 beam line at Shanghai Synchrotron Radiation Facility using Area Detector Systems Corporation Quantum 315r. The initial diffraction data sets were processed by the HKL3000 program (
      • Minor W.
      • Cymborowski M.
      • Otwinowski Z.
      • Chruszcz M.
      HKL-3000: The integration of data reduction and structure solution--from diffraction images to an initial model in minutes.
      ). AlAXEase structure was determined by molecular replacement using the SeMet-AlAXEase structure as the starting model. The refinement of AlAXEase structure was performed using Coot (
      • Emsley P.
      • Cowtan K.
      Coot: Model-building tools for molecular graphics.
      ) and Phenix (
      • Adams P.D.
      • Grosse-Kunstleve R.W.
      • Hung L.W.
      • Ioerger T.R.
      • McCoy A.J.
      • Moriarty N.W.
      • Read R.J.
      • Sacchettini J.C.
      • Sauter N.K.
      • Terwilliger T.C.
      PHENIX: Building new software for automated crystallographic structure determination.
      ). All structure figures were processed using PyMOL.

      DLS and CD spectroscopy

      The DLS experiments of AlAXEase protein and its mutants were carried out using DynaPro NanoStar (Wyatt Technology). The protein concentration was 1 mg/ml (10 mM Tris HCl buffer, pH 8.0, 100 mM NaCl). Data analysis was performed with the Dynamics 7.1.0 software.
      CD spectra of WT AlAXEase and its mutants were recorded at 25 °C on a J-1000 spectropolarimeter (JASCO). All the spectra were collected from 200 to 250 nm at a scanning rate of 200 nm/min with a path length of 0.1 cm. The protein concentration was 0.1 mg/ml. The thermal unfolding curves were recorded using the spectropolarimeter equipped with a CTU-100 temperature control unit (JASCO). The signal was recorded at 222 nm with a bandwidth of 1 nm. The temperature was monitored using an internal sensor, and the heating rate was 1 °C per min. A 0.1-cm path length cell was used. The protein concentration was 0.2 mg/ml.

      Fluorescence measurements

      Steady-state fluorescence measurements were performed using an FP-6500 spectrofluorometer (JASCO) equipped with a JULABO computer-controlled thermostat. The excitation wavelength was set at 280 nm and the emission wavelengths at 300 to 500 nm, respectively. Both excitation and emission bandwidths were 5 nm. Cuvettes with a 1-cm path length were used. Proteins were at a concentration of ∼0.06 mg/ml in 50 mM Tris HCl buffer (pH 8.0). Fluorescence spectra of AlAXEase and its mutants after incubation at different temperatures for 1 h were recorded, respectively.

      MD simulations

      The MD simulations of WT AlAXEase and its mutant E190A were conducted by using software package GROMACS 2019.6 (
      • Abraham M.J.
      • Murtola T.
      • Schulz R.
      • Páll S.
      • Smith J.C.
      • Hess B.
      • Lindahl E.
      GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.
      ), with the force field Amber99sb-ildn (
      • Hornak V.
      • Abel R.
      • Okur A.
      • Strockbine B.
      • Roitberg A.
      • Simmerling C.
      Comparison of multiple Amber force fields and development of improved protein backbone parameters.
      ) adopted. The enzyme structure was first placed into the center of a virtual cubic box with side length of 7.57 nm for WT and 7.36 nm for E190A and then solvated with 12,613 and 11,875 TIP3P water molecule model for WT and E190A, respectively. Sodium ions were added to the virtual water box as counter ions to neutralize the negative charge of the entire system (5 Na+ for WT and 4 Na+ for E190A). Energy minimization of the system was conducted using the steepest descent algorithm for 10,000 steps, followed by a 1-ns equilibration simulation with harmonic position restraints on the heavy atoms of protein to equilibrate the solvent molecules around the protein at the desired temperature. Subsequently, the simulation was performed for 200 ns at the target temperature without any position restraints. All simulations were performed under the NPT ensemble with periodic boundary conditions and a time step of 2 fs. The system was kept at a certain temperature using the v-rescale method, as well as the pressure was kept at 1 bar using the Parrinello–Rahman method. The temperature of the simulation was set to 280 K and 400 K. The final frame of the simulation performed under 400 K was used as the initial conformation to conduct another simulation under 280 K. According to the plot of the RMSD, trajectories that reached the equilibrium state (100 ns–200 ns) were used for analysis. The dynamics changes of the root mean square fluctuation values and the secondary structure against time were analyzed by using the built-in tools of GROMACS.

      Data availability

      The atomic coordinates and structure factors of AlAXEase have been deposited in the PDB with accession code 7DDY.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank the staffs from BL17U1 and BL18U1 beamlines of the National Facility for Protein Science Shanghai and Shanghai Synchrotron Radiation Facility, for assistance during data collection.
      We would like to thank Jingyao Qu, Jing Zhu, and Zhifeng Li from State Key laboratory of Microbial Technology of Shandong University for help and guidance in dynamic light scattering.
      This work was supported by the National Key Research and Development Program of China (2018YFC1406700, 2018YFC1406704), the National Natural Science Foundation of China (31630012, U1706207, 91851205, 91751101, 41906195, and 41676180), Major Scientific and Technological Innovation Project (MSTIP) of Shandong Province (2019JZZY010817), the Program of Shandong for Taishan Scholars (tspd20181203), and the Young Scholars Program of Shandong University (2017WLJH57).

      Author contributions

      Y. Z., W.-X. J., and J.-P. W. methodology; Y. Z. writing–original draft; H.-T. D., H.-Y. C., and C.-Y. L. software; H.-T. D., H.-Y. C., and C.-Y. L. formal analysis; X. Z., F. H., X.-Y. Z., X.-L. C., Y.-Z. Z., and P.-Y. L. project administration; X.-L. C. and P.-Y. L. writing–review and editing; Y.-Z. Z. and P.-Y. L. funding acquisition.

      Supporting information

      References

        • Adesioye F.A.
        • Makhalanyane T.P.
        • Biely P.
        • Cowan D.A.
        Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.
        Enzyme Microb. Technol. 2016; 93-94: 79-91
        • Komiya D.
        • Hori A.
        • Ishida T.
        • Igarashi K.
        • Samejima M.
        • Koseki T.
        • Fushinobu S.
        Crystal structure and substrate specificity modification of acetyl xylan esterase from Aspergillus luchuensis.
        Appl. Environ. Microbiol. 2017; 83e01251-17
        • Lombard V.
        • Golaconda Ramulu H.
        • Drula E.
        • Coutinho P.M.
        • Henrissat B.
        The carbohydrate-active enzymes database (CAZy) in 2013.
        Nucleic Acids Res. 2014; 42: D490-D495
        • Alalouf O.
        • Balazs Y.
        • Volkinshtein M.
        • Grimpel Y.
        • Shoham G.
        • Shoham Y.
        A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
        J. Biol. Chem. 2011; 286: 41993-42001
        • Soni S.
        • Sathe S.S.
        • Odaneth A.A.
        • Lali A.M.
        • Chandrayan S.K.
        SGNH hydrolase-type esterase domain containing Cbes-AcXE2: A novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.
        Extremophiles. 2017; 21: 687-697
        • Koutaniemi S.
        • van Gool M.P.
        • Juvonen M.
        • Jokela J.
        • Hinz S.W.
        • Schols H.A.
        • Tenkanen M.
        Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation.
        J. Biotechnol. 2013; 168: 684-692
        • Tian Q.
        • Song P.
        • Jiang L.
        • Li S.
        • Huang H.
        A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid.
        Appl. Microbiol. Biotechnol. 2014; 98: 2081-2089
        • Park S.H.
        • Yoo W.
        • Lee C.W.
        • Jeong C.S.
        • Shin S.C.
        • Kim H.W.
        • Park H.
        • Kim K.K.
        • Kim T.D.
        • Lee J.H.
        Crystal structure and functional characterization of a cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp.
        PLoS One. 2018; 13e0206260
        • Razeq F.M.
        • Jurak E.
        • Stogios P.J.
        • Yan R.
        • Tenkanen M.
        • Kabel M.A.
        • Wang W.
        • Master E.R.
        A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans.
        Biotechnol. Biofuels. 2018; 11: 74
        • Akoh C.C.
        • Lee G.C.
        • Liaw Y.C.
        • Huang T.H.
        • Shaw J.F.
        GDSL family of serine esterases/lipases.
        Prog. Lipid Res. 2004; 43: 534-552
        • Li J.
        • Derewenda U.
        • Dauter Z.
        • Smith S.
        • Derewenda Z.S.
        Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.
        Nat. Struct. Biol. 2000; 7: 555-559
        • Molgaard A.
        • Kauppinen S.
        • Larsen S.
        Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases.
        Structure. 2000; 8: 373-383
        • Lescic Asler I.
        • Stefanic Z.
        • Marsavelski A.
        • Vianello R.
        • Kojic-Prodic B.
        Catalytic dyad in the SGNH hydrolase superfamily: In-depth insight into structural parameters tuning the catalytic process of extracellular lipase from Streptomyces rimosus.
        ACS Chem. Biol. 2017; 12: 1928-1936
        • Correia M.A.S.
        • Prates J.A.M.
        • Bras J.
        • Fontes C.M.G.A.
        • Newman J.A.
        • Lewis R.J.
        • Gilbert H.J.
        • Flint J.E.
        Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition.
        J. Mol. Biol. 2008; 379: 64-72
        • Lansky S.
        • Alalouf O.
        • Solomon H.V.
        • Alhassid A.
        • Govada L.
        • Chayen N.E.
        • Belrhali H.
        • Shoham Y.
        • Shoham G.
        A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.
        Acta Crystallogr. D Biol. Crystallogr. 2014; 70: 261-278
        • Till M.
        • Goldstone D.C.
        • Attwood G.T.
        • Moon C.D.
        • Kelly W.J.
        • Arcus V.L.
        Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus.
        Proteins. 2013; 81: 911-917
        • Martinez-Martinez I.
        • Navarro-Fernandez J.
        • Daniel Lozada-Ramirez J.
        • Garcia-Carmona F.
        • Sanchez-Ferrer A.
        YesT: A new rhamnogalacturonan acetyl esterase from Bacillus subtilis.
        Proteins. 2008; 71: 379-388
        • Blum D.L.
        • Li X.L.
        • Chen H.Z.
        • Ljungdahl L.G.
        Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.
        Appl. Environ. Microbiol. 1999; 65: 3990-3995
        • Nagel Z.D.
        • Cun S.
        • Klinman J.P.
        Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases.
        J. Biol. Chem. 2013; 288: 14087-14097
        • Papaleo E.
        • Riccardi L.
        • Villa C.
        • Fantucci P.
        • De Gioia L.
        Flexibility and enzymatic cold-adaptation: A comparative molecular dynamics investigation of the elastase family.
        Biochim. Biophys. Acta. 2006; 1764: 1397-1406
        • Siddiqui K.S.
        • Cavicchioli R.
        Cold-adapted enzymes.
        Annu. Rev. Biochem. 2006; 75: 403-433
        • Xie B.B.
        • Bian F.
        • Chen X.L.
        • He H.L.
        • Guo J.
        • Gao X.
        • Zeng Y.X.
        • Chen B.
        • Zhou B.C.
        • Zhang Y.Z.
        Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and Arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics new insights into relationship between conformational flexibility and hydrogen bonding.
        J. Biol. Chem. 2009; 284: 9257-9269
        • Leiros H.K.
        • Pey A.L.
        • Innselset M.
        • Moe E.
        • Leiros I.
        • Steen I.H.
        • Martinez A.
        Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.
        J. Biol. Chem. 2007; 282: 21973-21986
        • Fedoy A.E.
        • Yang N.
        • Martinez A.
        • Leiros H.K.
        • Steen I.H.
        Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.
        J. Mol. Biol. 2007; 372: 130-149
        • Li D.D.
        • Peng M.
        • Wang N.
        • Wang X.J.
        • Zhang X.Y.
        • Chen X.L.
        • Su H.N.
        • Zhang Y.Z.
        • Shi M.
        Arcticibacterium luteifluviistationis gen. nov., sp. nov., isolated from Arctic seawater.
        Int. J. Syst. Evol. Microbiol. 2017; 67: 664-669
        • Li Y.
        • Guo X.H.
        • Dang Y.R.
        • Sun L.L.
        • Zhang X.Y.
        • Chen X.L.
        • Qin Q.L.
        • Wang P.
        Complete genome sequence of Arcticibacterium luteifluviistationis SM1504T, a cytophagaceae bacterium isolated from Arctic surface seawater.
        Stand Genomic Sci. 2018; 13: 33
        • Pfeffer J.M.
        • Weadge J.T.
        • Clarke A.J.
        Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.
        J. Biol. Chem. 2013; 288: 2605-2613
        • Tang M.A.
        • Motoshima H.
        • Watanabe K.
        Fluorescence studies on the stability, flexibility and substrate-induced conformational changes of acetate kinases from psychrophilic and mesophilic bacteria.
        Protein J. 2012; 31: 337-344
        • Struvay C.
        • Feller G.
        Optimization to low temperature activity in psychrophilic enzymes.
        Int. J. Mol. Sci. 2012; 13: 11643-11665
        • Sanchez-Ruiz J.M.
        • Makhatadze G.I.
        To charge or not to charge?.
        Trends Biotechnol. 2001; 19: 132-135
        • Tanaka Y.
        • Tsumoto K.
        • Yasutake Y.
        • Umetsu M.
        • Yao M.
        • Fukada H.
        • Tanaka I.
        • Kumagai I.
        How oligomerization contributes to the thermostability of an Archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii.
        J. Biol. Chem. 2004; 279: 32957-32967
        • Ogasahara K.
        • Ishida M.
        • Yutani K.
        Stimulated interaction between alpha and beta subunits of tryptophan synthase from hyperthermophile enhances its thermal stability.
        J. Biol. Chem. 2003; 278: 8922-8928
        • Martinez L.
        Automatic identification of mobile and rigid substructures in molecular dynamics simulations and fractional structural fluctuation analysis.
        PLoS One. 2015; 10e0119264
        • Zhao F.
        • Cao H.Y.
        • Zhao L.S.
        • Zhang Y.
        • Li C.Y.
        • Zhang Y.Z.
        • Li P.Y.
        • Wang P.
        • Chen X.L.
        A novel subfamily of endo-beta-1,4-glucanases in glycoside hydrolase family 10.
        Appl. Environ. Microbiol. 2019; 85e01029-19
        • Rutkiewicz M.
        • Bujacz A.
        • Bujacz G.
        Structural features of cold-adapted dimeric GH2 beta-D-galactosidase from Arthrobacter sp. 32cB.
        Biochim. Biophys. Acta Proteins Proteom. 2019; 1867: 776-786
        • Lee C.W.
        • Kwon S.
        • Park S.H.
        • Kim B.Y.
        • Yoo W.
        • Ryu B.H.
        • Kim H.W.
        • Shin S.C.
        • Kim S.
        • Park H.
        • Kim T.D.
        • Lee J.H.
        Crystal structure and functional characterization of an esterase (EaEST) from Exiguobacterium antarcticum.
        PLoS One. 2017; 12e0169540
        • Le L.T.H.L.
        • Yoo W.
        • Jeon S.
        • Lee C.
        • Kim T.D.
        Biodiesel and flavor compound production using a novel promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from the psychrophilic bacterium Halocynthiibacter arcticus.
        Biotechnol. Biofuels. 2020; 13: 55
        • Lian K.
        • Leiros H.K.
        • Moe E.
        MutT from the fish pathogen Aliivibrio salmonicida is a cold-active nucleotide-pool sanitization enzyme with unexpectedly high thermostability.
        FEBS Open Bio. 2015; 5: 107-116
        • Yang G.
        • De Santi C.
        • de Pascale D.
        • Pucciarelli S.
        • Pucciarelli S.
        • Miceli C.
        Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus.
        Biochimie. 2013; 95: 1795-1806
        • Kulakova L.
        • Galkin A.
        • Nakayama T.
        • Nishino T.
        • Esaki N.
        Cold-active esterase from Psychrobacter sp. Ant300: Gene cloning, characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability.
        Biochim. Biophys. Acta. 2004; 1696: 59-65
        • Liu H.
        • Naismith J.H.
        An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.
        BMC Biotechnol. 2008; 8: 91
        • Li P.Y.
        • Ji P.
        • Li C.Y.
        • Zhang Y.
        • Wang G.L.
        • Zhang X.Y.
        • Xie B.B.
        • Qin Q.L.
        • Chen X.L.
        • Zhou B.C.
        • Zhang Y.Z.
        Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family.
        J. Biol. Chem. 2014; 289: 19031-19041
        • Minor W.
        • Cymborowski M.
        • Otwinowski Z.
        • Chruszcz M.
        HKL-3000: The integration of data reduction and structure solution--from diffraction images to an initial model in minutes.
        Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 859-866
        • Emsley P.
        • Cowtan K.
        Coot: Model-building tools for molecular graphics.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132
        • Adams P.D.
        • Grosse-Kunstleve R.W.
        • Hung L.W.
        • Ioerger T.R.
        • McCoy A.J.
        • Moriarty N.W.
        • Read R.J.
        • Sacchettini J.C.
        • Sauter N.K.
        • Terwilliger T.C.
        PHENIX: Building new software for automated crystallographic structure determination.
        Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1948-1954
        • Abraham M.J.
        • Murtola T.
        • Schulz R.
        • Páll S.
        • Smith J.C.
        • Hess B.
        • Lindahl E.
        GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers.
        SoftwareX. 2015; 1-2: 19-25
        • Hornak V.
        • Abel R.
        • Okur A.
        • Strockbine B.
        • Roitberg A.
        • Simmerling C.
        Comparison of multiple Amber force fields and development of improved protein backbone parameters.
        Proteins. 2006; 65: 712-725
        • Li P.Y.
        • Chen X.L.
        • Ji P.
        • Li C.Y.
        • Wang P.
        • Zhang Y.
        • Xie B.B.
        • Qin Q.L.
        • Su H.N.
        • Zhou B.C.
        • Zhang Y.Z.
        • Zhang X.Y.
        Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.
        J. Biol. Chem. 2015; 290: 11188-11198
        • Wang P.
        • Chen X.L.
        • Li C.Y.
        • Gao X.
        • Zhu D.Y.
        • Xie B.B.
        • Qin Q.L.
        • Zhang X.Y.
        • Su H.N.
        • Zhou B.C.
        • Xun L.Y.
        • Zhang Y.Z.
        Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial dimethylsulfoniopropionate lyase: A new enzyme from an old fold.
        Mol. Microbiol. 2015; 98: 289-301