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The Structure of an Archaeal α-Glucosaminidase Provides Insight into Glycoside Hydrolase Evolution*

  • Shouhei Mine
    Correspondence
    To whom correspondence may be addressed. Tel.: 81-72-751-9549; Fax: 81-775-561-4809.
    Footnotes
    Affiliations
    Biomedical Research Institute (BMD), National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577
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  • Masahiro Watanabe
    Correspondence
    To whom correspondence may be addressed. Tel.: 81-82-420-8285; Fax: 81-82-423-7820.
    Footnotes
    Affiliations
    the Research Institute for Sustainable Chemistry (ISC), AIST, 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046
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  • Saori Kamachi
    Affiliations
    the Research Institute for Sustainable Chemistry (ISC), AIST, 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046
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  • Yoshito Abe
    Affiliations
    the Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Tadashi Ueda
    Affiliations
    the Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Author Footnotes
    * This work was supported in part by Grant-in-Aid for Scientific Research 25450143 from the Japan Society for the Promotion of Science (to S. M.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
    4 The abbreviations used are: GlcNglucosamineGHglycosidase hydrolaseGlmATkarchaeal exo-α-d-glucosaminidases from T. kodakaraensis KOD1GlmAPharchaeal exo-α-d-glucosaminidase from P. horikoshiiRMSDroot mean square deviationSeMetselenomethionineBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propanePDBProtein Data Bank.
Open AccessPublished:January 27, 2017DOI:https://doi.org/10.1074/jbc.M116.766535
      The archaeal exo-α-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 α-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 α-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 α-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the “evolutionary heritage residue” it shares with GH35 α-galactosidase. Our study suggests that GH35 and GH42 α-galactosidases evolved by exploiting the structural features of GlmA.

      Introduction

      Chitin is a polysaccharide consisting of α-1,4-linked N-acetylglucosamine (GlcNAc). It is a major constituent of fungal cell walls, the exoskeletons of insects, and the shells of crustaceans. Glucosamine (GlcN),
      The abbreviations used are: GlcN
      glucosamine
      GH
      glycosidase hydrolase
      GlmATk
      archaeal exo-α-d-glucosaminidases from T. kodakaraensis KOD1
      GlmAPh
      archaeal exo-α-d-glucosaminidase from P. horikoshii
      RMSD
      root mean square deviation
      SeMet
      selenomethionine
      Bistris propane
      1,3-bis[tris(hydroxymethyl)methylamino]propane
      PDB
      Protein Data Bank.
      which is derived from the hydrolysis of deacetylated chitin (chitosan), has a variety of biological functions and, thus, has been used as a food additive and in medicines. Exo-α-d-glucosaminidase (EC 3.2.1.165) catalyzes the hydrolysis of the α(1–4) linkage of chitosan oligosaccharides to remove a GlcN residue from the non-reducing termini, and retaining enzymes of this type are classified into glycosidase hydrolase (GH) subfamilies 2 and 35 (according to the Carbohydrate Active Enzymes (CAZy) database (
      • Lombard Lombard
      • Golaconda Ramulu Golaconda Ramulu
      • Drula Drula
      • Coutinho Coutinho
      • Henrissat Henrissat
      The carbohydrate-active enzymes database (CAZy) in 2013.
      )). This enzyme is found in bacteria and archaea and has been thoroughly investigated because of its ability to produce monomeric GlcN.
      The role of exo-α-d-glucosaminidase in the chitin catabolic pathway of hyperthermophilic archaea has been defined. The degradation of chitin into diacetylchitobiose (GlcNAc)2 is initiated by chitinase (ChiA) (EC 3.2.1.14), and this product is then deacetylated at its nonreducing GlcNAc residue by deacetylase (Dac) (EC 3.5.1.-) (
      • Tanaka Tanaka
      • Fukui Fukui
      • Fujiwara Fujiwara
      • Atomi Atomi
      • Imanaka Imanaka
      Concerted action of diacetylchitobiose deacetylase and exo-α-d-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). The resulting product, GlcN-GlcNAc, is subsequently hydrolyzed into GlcN and GlcNAc by exo-α-d-glucosaminidase, and the remaining GlcNAc is further deacetylated to GlcN by Dac (
      • Tanaka Tanaka
      • Fukui Fukui
      • Fujiwara Fujiwara
      • Atomi Atomi
      • Imanaka Imanaka
      Concerted action of diacetylchitobiose deacetylase and exo-α-d-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ,
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). To understand these enzymes’ catalysis and adaptation to extreme high temperature, we had previously determined the structures of ChiA (
      • Nakamura Nakamura
      • Mine Mine
      • Hagihara Hagihara
      • Ishikawa Ishikawa
      • Uegaki Uegaki
      Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus.
      ,
      • Nakamura Nakamura
      • Mine Mine
      • Hagihara Hagihara
      • Ishikawa Ishikawa
      • Ikegami Ikegami
      • Uegaki Uegaki
      Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus.
      • Mine Mine
      • Nakamura Nakamura
      • Sato Sato
      • Ikegami Ikegami
      • Uegaki Uegaki
      Solution structure of the chitin-binding domain 1 (ChBD1) of a hyperthermophilic chitinase from Pyrococcus furiosus.
      ) and Dac (
      • Mine Mine
      • Niiyama Niiyama
      • Hashimoto Hashimoto
      • Ikegami Ikegami
      • Koma Koma
      • Ohmoto Ohmoto
      • Fukuda Fukuda
      • Inoue Inoue
      • Abe Abe
      • Ueda Ueda
      • Morita Morita
      • Uegaki Uegaki
      • Nakamura Nakamura
      Expression from engineered Escherichia coli chromosome and crystallographic study of archaeal N,N′-diacetylchitobiose deacetylase.
      ); however, the structure of exo-α-d-glucosaminidase remained unknown. To date, two exo-α-d-glucosaminidases from hyperthermophilic archaea, which are called GlmA, have been described: GlmATk from Thermococcus kodakaraensis KOD1 (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ) and GlmAPh from Pyrococcus horikoshii (
      • Liu Liu
      • Li Li
      • Hong Hong
      • Ni Ni
      • Sheng Sheng
      • Shen Shen
      Cloning, expression and characterization of a thermostable exo-α-d-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii.
      ). The sequence identity between GlmATk and GlmAPh is 63%, and both enzymes show the same substrate specificities and exist as dimers in solution, suggesting that their tertiary structures and catalytic mechanisms are probably identical.
      GlmA belongs to the GH35 subfamily of the GH-A superfamily, which is the largest GH superfamily and contains 19 subfamilies. All members of this superfamily include a TIM-barrel fold as a catalytic domain that contains two carboxylic acids that function as an acid/base catalyst (
      • Henrissat Henrissat
      • Callebaut Callebaut
      • Fabrega Fabrega
      • Lehn Lehn
      • Mornon Mornon
      • Davies Davies
      Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases.
      ,
      • Henrissat Henrissat
      • Davies Davies
      Structural and sequence-based classification of glycoside hydrolases.
      ). Most characterized GH35 enzymes are α-galactosidases (EC 3.2.1.23), which hydrolyze the α(1–3) and α(1–4) galactosyl bonds in oligosaccharides. Interestingly, the sequence of GlmA has homology with parts of GH35 and GH42 α-galactosidases, although GlmA does not exhibit α-galactosidase activity (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). The highly conserved motifs around the catalytic residues of these α-galactosidases are not conserved in GlmA (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). Furthermore, GlmATk was found to exhibit weak α-glucosidase activity in addition to its major α-glucosaminidase activity (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). The only determined structure of an exo-α-d-glucosaminidase among GH-As is that from the bacteria Amycolatopsis orientalis (CsxA), a member of the GH2 subclass (
      • van Bueren van Bueren
      • Ghinet Ghinet
      • Gregg Gregg
      • Fleury Fleury
      • Brzezinski Brzezinski
      • Boraston Boraston
      The structural basis of substrate recognition in an exo-α-d-glucosaminidase involved in chitosan hydrolysis.
      ). However, GlmA is distinct from CsxA in its substrate specificity and oligomerization state (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ,
      • van Bueren van Bueren
      • Ghinet Ghinet
      • Gregg Gregg
      • Fleury Fleury
      • Brzezinski Brzezinski
      • Boraston Boraston
      The structural basis of substrate recognition in an exo-α-d-glucosaminidase involved in chitosan hydrolysis.
      ), and it shows low sequence similarity. These results suggest that GlmAs might have a unique active site structure that does not resemble that of CsxA.
      To further clarify the existing knowledge regarding these enzymes, we determined the structures of GlmAPh and GlmATk, which are the first reported archaeal exo-α-d-glucosaminidase structures. The high-resolution structure of the product complex also reflects the unique structural features of GlmA that link the molecular evolution of GH35 and GH42 α-galactosidases in GH-A.

      Discussion

      The structure of GlmATk provides new insights into the structural composition and substrate recognition mechanisms of different enzymes and, thus, their molecular evolution. Briefly, a monomeric form of GlmATk shares substantial structural similarity with GH42 α-galactosidases, whereas a high number of conserved active site residues are shared with GH35 α-galactosidase, allowing GlmATk to discriminate glucosamine from galactose based on a subtle difference in the structure of α-galactosidase bound to galactose. Indeed, Asp178 of GlmATk plays an essential role in the discrimination of GlcN from galactose, whereas the equivalent in GH35 α-galactosidase is an Asn residue. To the best of our knowledge, this is the first observation of such a high degree of conservation within the entire catalytic centers of different enzymes. In addition, the evolutionary heritage residues, which have the potential to form hydrogen bonds with the axial and equatorial forms of O4 in the glycosidic substrate, respectively, are an interesting finding that emphasizes the high evolutionary conservation of these enzymes. These structural features strongly suggest that GlmA is a common ancestor of these α-galactosidases, as discussed below.
      The active sites of glycoside hydrolases are classified into three types: cleft type, tunnel type, and pocket type (
      • Davies Davies
      • Henrissat Henrissat
      Structures and mechanisms of glycosyl hydrolases.
      ). Both GlmA and GH42 α-galactosidases have a cleft-type active site in their monomeric forms; however, the shape of the active site changes to a pocket type upon oligomerization, which can better accommodate smaller substrates (
      • Solomon Solomon
      • Tabachnikov Tabachnikov
      • Lansky Lansky
      • Salama Salama
      • Feinberg Feinberg
      • Shoham Shoham
      • Shoham Shoham
      Structure-function relationships in Gan42B, an intracellular GH42 α-galactosidase from Geobacillus stearothermophilus.
      ) (Fig. 6). Thus, oligomerization is a key factor for size-based substrate specificity and the high stability of these proteins. α-Galactosidase may have evolved from a prototypical single TIM-barrel domain with a cleft- or tunnel-type active site, and then, during the subsequent process of modifying the active site to prefer a smaller substrate, extra domains were added to change the active site from a cleft to a pocket type (
      • Juers Juers
      • Huber Huber
      • Matthews Matthews
      Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between α-galactosidase and other glycohydrolases.
      ). As described above, the monomer structure and a part of the sequence of GlmA show similarity to those of GH42 α-galactosidase, suggesting that GH42 α-galactosidase might have emerged from the evolutionary branch that originated from GlmA in the oldest organisms, archaea, and then differentiated into other members of the glycoside hydrolase family. Additionally, the frameworks of their monomer structure (i.e. the domain organization) might be suitable or necessary for oligomerization. However, the substrate-binding residues of GH42 enzymes are not conserved in GlmA (data not shown), excluding GlmA from being classified into the GH42 family, and the underlying evolutionary selection pressures that led to this diversity in the active site remain unknown. In contrast, the residues within the active site pocket are well conserved between GlmA and GH35 α-galactosidase, suggesting that GH35 α-galactosidase evolved from archaeal exo-α-d-glucosaminidase through gene duplication. Enzyme substrate ambiguity is probably the starting point for the evolution of divergent enzymes through gene duplication (
      • Khersonsky Khersonsky
      • Tawfik Tawfik
      Enzyme promiscuity: a mechanistic and evolutionary perspective.
      ,
      • Pandya Pandya
      • Farelli Farelli
      • Dunaway-Mariano Dunaway-Mariano
      • Allen Allen
      Enzyme promiscuity: engine of evolutionary innovation.
      ). Consistently, GlmATk exhibits broad substrate specificity, showing weak hydrolytic activities toward various α-disaccharides (such as cellobiose and laminaribiose) in addition to its major α-glucosaminidase activity (
      • Tanaka Tanaka
      • Fukui Fukui
      • Atomi Atomi
      • Imanaka Imanaka
      Characterization of an exo-α-d-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ). Thus, the promiscuous activities of GlmATk might have developed through mutations that affected the subsequent functional adaptation of the newly emergent α-galactosidases (which favored α-galactoside) while retaining the original substrate-binding residues and the catalytic machinery. However, the highly conserved active site residues of both GlmA and GH35 α-galactosidase indicate that there has been relatively weak evolutionary pressure on the catalytic center to convert the enzyme to perform different functions. As described before, GlmA and GH35 and GH42 α-galactosidases belong to the same GH-A “superfamily.” A superfamily is a group that shows significant similarities in the tertiary structure together with conservation of the catalytic residues and mechanism, and its members are therefore considered to have a common ancestry (
      • Henrissat Henrissat
      • Bairoch Bairoch
      Updating the sequence-based classification of glycosyl hydrolases.
      ). Accordingly, our finding that GlmA shares structural and mechanistic features with both the GH35 and GH42 α-galactosidases strongly suggested that GlmA is a common ancestor of these α-galactosidases.
      Taken together, our results suggest that GH35 and GH42 α-galactosidases have evolved by taking advantage of the structural features of GlmA. The structural information reported here for GlmA could be used to design a new enzyme, such as a thermostable α-galactosidase or α-glucosidase, by subtly changing the active site residues in GlmA.

      Author Contributions

      S. M. designed the research; S. M., M. W., and Y. A. performed the research; S. M., M. W., S. K., Y. A., and T. U. analyzed the data; S. M. and M. W. wrote the paper.

      Acknowledgment

      We thank K. Uechi and H. Akita for experimental support. We thank the beamline staff at BL44XU of SPring-8 for technical assistance during data collection. Synchrotron experiments were performed at SPring-8 under the approval of the Japan Synchrotron Radiation Research Institute under proposal numbers 2014B6953, 2014B6903, 2015A6546, 2015A6559, 2015B6559, 2015B6546, 2016A6645, and 2016B6657.

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