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Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus*

  • Yejun Han
    Footnotes
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801
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  • Vinayak Agarwal
    Footnotes
    Affiliations
    Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801

    Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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  • Dylan Dodd
    Footnotes
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Department of Microbiology, University of Illinois, Urbana, Illinois 61801
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  • Jason Kim
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Department of Molecular and Cellular Biology, University of Illinois, Urbana, Illinois 61801
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  • Brian Bae
    Affiliations
    Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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  • Roderick I. Mackie
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801
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  • Satish K. Nair
    Correspondence
    To whom correspondence may be addressed: Dept. of Biochemistry, 600 S. Matthews Ave., University of Illinois, Urbana, IL 61801. Tel.: 217-333-0641; Fax: 217-244-5858
    Affiliations
    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801

    Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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  • Isaac K.O. Cann
    Correspondence
    To whom correspondence may be addressed: Energy Biosciences Institute, 1105 Institute for Genomic Biology, 1206 West Gregory Dr., University of Illinois, Urbana, IL 61801. Tel.: 217-333-2090; Fax: 217-333-8286
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Department of Microbiology, University of Illinois, Urbana, Illinois 61801

    Department of Molecular and Cellular Biology, University of Illinois, Urbana, Illinois 61801

    Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801
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  • Author Footnotes
    * This work was supported by the Energy Biosciences Institute.
    This article contains supplemental Experimental Procedures, Figs. S1–S3, Tables S1 and S2, and additional references.
    1 These authors contributed equally to this work.
Open AccessPublished:August 22, 2012DOI:https://doi.org/10.1074/jbc.M112.391532
      Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

      Introduction

      Hemicellulose, one of the main components of the plant cell wall, is one of the most abundant polysaccharides in nature. The efficient degradation of the polymer has gained increasing interest due to the capacity to convert its monomeric sugars to bioenergy products such as ethanol (
      • Dodd D.
      • Cann I.K.
      Enzymatic deconstruction of xylan for biofuel production.
      ). Xylan, the most common hemicellulose, is a heterogeneous polysaccharide composed mostly of linear chains of xylose with side chain substitutions. The backbone of xylan is composed of β-1,4-linked d-xylopyranosyl units and may be decorated with 4-O-methyl-d-glucuronyl, l-arabinofuranosyl, and acetyl substituents (
      • Yeoman C.J.
      • Han Y.
      • Dodd D.
      • Schroeder C.M.
      • Mackie R.I.
      • Cann I.K.
      Thermostable enzymes as biocatalysts in the biofuel industry.
      ). The complete degradation of xylan requires the synergistic activity of several hemicellulolytic enzymes, such as β-1,4-endoxylanase, β-xylosidase, α-glucuronidase, α-l-arabinosidase, and acetylxylan esterase (
      • Dodd D.
      • Cann I.K.
      Enzymatic deconstruction of xylan for biofuel production.
      ). To facilitate a concerted action of these enzymes for hemicellulose degradation, several microorganisms have evolved gene clusters encoding the different hemicellulolytic enzymes (
      • Dodd D.
      • Cann I.K.
      Enzymatic deconstruction of xylan for biofuel production.
      ,
      • Baba T.
      • Shinke R.
      • Nanmori T.
      Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, β-xylosidase and xylanase, of Bacillus stearothermophilus 21.
      ,
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4. Sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and β-(1,4)-xylosidase activities.
      ,
      • Jun H.S.
      • Ha J.K.
      • Malburg Jr., L.M.
      • Verrinder G.A.
      • Forsberg C.W.
      Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85.
      ,
      • Shulami S.
      • Gat O.
      • Sonenshein A.L.
      • Shoham Y.
      The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.
      ). The transport mechanism for xylan degradation products has been fairly well described in bacteria such as Streptomyces lividans and Geobacillus stearothermophilus (
      • Hurtubise Y.
      • Shareck F.
      • Kluepfel D.
      • Morosoli R.
      A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins.
      ,
      • Shulami S.
      • Zaide G.
      • Zolotnitsky G.
      • Langut Y.
      • Feld G.
      • Sonenshein A.L.
      • Shoham Y.
      A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.
      ). However, our knowledge in this area of sugar metabolism by bacteria is still limited.
      β-Xylosidases and α-glucuronidases are two critical enzymes for xylan hydrolysis. Endoxylanases cleave xylan polysaccharides into xylo-oligosaccharides that may be decorated with methylglucuronic acids and hence impede the effectiveness of the β-xylosidase, the enzyme responsible for cleavage of xylose monomers from xylo-oligosaccharides (
      • Cullen D.
      • Kersten P.
      ). Microorganisms have therefore developed the enzymatic activity to remove the methylglucuronic acid side chains. Thus, α-glucuronidases cleave the α-1,2-glycosidic bond between 4-O-methyl α-glucuronic acid and the xylopyranosyl unit of xylo-oligosaccharides (
      • Mierzwa M.
      • Tokarzewska-Zadora J.
      • Deptu̸a T.
      • Rogalski J.
      • Szczodrak J.
      Purification and characterization of an extracellular α-d-glucuronidase from Phlebia radiata.
      ). To date, most of the cloned microbial β-xylosidases fall within glycoside hydrolase (GH)
      The abbreviations used are: GH
      glycoside hydrolase
      MGX
      (4-O-methyl-α-d-glucurono)-d-xylan
      BWX
      birchwood xylan
      pNP
      para-nitrophenyl
      ITC
      isothermal titration calorimetry
      PDB
      Protein Data Bank
      r.m.s.d.
      root mean square deviation
      SLH
      surface layer homology
      X1
      monomeric xylose
      pNPGlu
      pNP-β-d-glucopyranoside
      pNPX
      pNP-β-d-xylopyranoside
      X2
      xylobiose
      X3
      xylotriose
      X4
      xylotetraose
      X5
      xylopentaose
      X6
      xylohexaose
      G2
      cellobiose
      G3
      cellotriose
      G4
      cellotetraose
      G5
      cellopentaose
      ABC
      ATP-binding cassette
      Q
      quantitative
      FnIII
      fibronectin repeat 3
      HPAEC-PAD
      high performance anion exchange chromatography-pulsed amperometric detection.
      families 3, 30, 39, 43, 51, 52, and 54 (
      • Yeoman C.J.
      • Han Y.
      • Dodd D.
      • Schroeder C.M.
      • Mackie R.I.
      • Cann I.K.
      Thermostable enzymes as biocatalysts in the biofuel industry.
      ,
      • Czjzek M.
      • Ben David A.
      • Bravman T.
      • Shoham G.
      • Henrissat B.
      • Shoham Y.
      Enzyme-substrate complex structures of a GH39 β-xylosidase from Geobacillus stearothermophilus.
      ,
      • Eneyskaya E.V.
      • Ivanen D.R.
      • Bobrov K.S.
      • Isaeva-Ivanova L.S.
      • Shabalin K.A.
      • Savel'ev A.N.
      • Golubev A.M.
      • Kulminskaya A.A.
      Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.
      ,
      • Rohman A.
      • van Oosterwijk N.
      • Kralj S.
      • Dijkhuizen L.
      • Dijkstra B.W.
      • Puspaningsih N.N.
      Purification, crystallization, and preliminary x-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08.
      ,
      • Shallom D.
      • Shoham Y.
      Microbial hemicellulases.
      ), whereas characterized α-glucuronidases are assigned to either GH family 67 (
      • Shulami S.
      • Gat O.
      • Sonenshein A.L.
      • Shoham Y.
      The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.
      ,
      • De Wet B.J.
      • Van Zyl W.H.
      • Prior B.A.
      Characterization of the Aureobasidium pullulans α-glucuronidase expressed in Saccharomyces cerevisiae.
      ,
      • Nagy T.
      • Nurizzo D.
      • Davies G.J.
      • Biely P.
      • Lakey J.H.
      • Bolam D.N.
      • Gilbert H.J.
      The α-glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants.
      ) or GH family 115 (
      • Ryabova O.
      • Vrsanská M.
      • Kaneko S.
      • van Zyl W.H.
      • Biely P.
      A novel family of hemicellulolytic α-glucuronidase.
      ).
      With a number of advantages over mesophilic enzymes, thermostable enzymes are especially thought to improve hydrolytic performance and overall economy of the process of biofuel production from the plant cell wall. Thermostable enzymes have thus been gaining increasing attention in the field of biofuels (
      • Maheshwari R.
      • Bharadwaj G.
      • Bhat M.K.
      Thermophilic fungi. Their physiology and enzymes.
      ,
      • Viikari L.
      • Alapuranen M.
      • Puranen T.
      • Vehmaanperä J.
      • Siika-Aho M.
      Thermostable enzymes in lignocellulose hydrolysis.
      ).
      Caldanaerobius polysaccharolyticus, an anaerobic thermophilic bacterium, was isolated in Illinois(
      • Cann I.K.
      • Stroot P.G.
      • Mackie K.R.
      • White B.A.
      • Mackie R.I.
      Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp. nov. Thermoanaerobacterium zeae sp. nov., and emendation of the genus Thermoanaerobacterium.
      ,
      • Lee Y.J.
      • Mackie R.I.
      • Cann I.K.
      • Wiegel J.
      Description of Caldanaerobius fijiensis gen. nov., sp. nov., an inulin-degrading, ethanol-producing, thermophilic bacterium from a Fijian hot spring sediment, and reclassification of Thermoanaerobacterium polysaccharolyticum Thermoanaerobacterium zeae as Caldanaerobius polysaccharolyticus comb. nov. Caldanaerobius zeae comb. nov.
      ). Several thermostable hemicellulolytic enzymes have been cloned and characterized from C. polysaccharolyticus (
      • Cann I.K.
      • Kocherginskaya S.
      • King M.R.
      • White B.A.
      • Mackie R.I.
      Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum.
      ,
      • King M.R.
      • White B.A.
      • Blaschek H.P.
      • Chassy B.M.
      • Mackie R.I.
      • Cann I.K.
      Purification and characterization of a thermostable α-galactosidase from Thermoanaerobacterium polysaccharolyticum.
      ). Recently, by determining the partial genome sequence of this bacterium, we have identified all of the genes encoding enzymes that will permit reconstitution of a hemicellulolytic enzyme mixture, a highly desirable product in the emerging biofuel industry, from C. polysaccharolyticus. These enzymes include a β-1,4-endoxylanase (Xyn10A), β-xylosidase (Xyl3A), α-glucuronidase (Agu67A), α-l-arabinofuranosidase (Ara51A), and an acetylxylan esterase (Axe4A).
      In this study, Xyl3A and Agu67A appeared to be components of a pentose sugar metabolism cluster and were cloned and characterized from C. polysaccharolyticus. Because the endoxylanase, Xyn10A, is not linked to this gene cluster, we hypothesized that the gene products from the cluster serve to capture nutrients (xylo-oligosaccharides) generated by Xyn10A for further hydrolysis to directly feed them into the pentose-phosphate pathway. Here, we express the recombinant form of each protein and demonstrate their contributions to xylan metabolism by C. polysaccharolyticus. We also identify a membrane-integral ATP-dependent sugar complex that likely transports the end products of xylan degradation into the cell. To determine the chain length preference for this transporter, we carried out biochemical analysis of the solute-binding component (XBP1) of the complex, and we solved the co-crystal structure of this polypeptide in complex with xylotriose. It is anticipated that the clustering of the genes involved in xylan utilization in this thermophilic bacterium will also offer an opportunity to transfer the phenotype to other organisms with tractable genetic systems for further engineering and improvement of xylan utilization.

      DISCUSSION

      Xyn10A is a multimodular endoxylanase composed of a GH 10 endoxylanase module flanked on the N terminus by a tandem repeat of CBM 22 and on the C terminus by another tandem repeat of CBM 9, followed by three SLH modules (Fig. 1A). This modular organization is conserved among six other bacteria, two of the genus Thermoanaerobacter and four of the genus Thermoanaerobacterium (supplemental Fig. 3). Beyond these organisms, there are hundreds of homologous proteins (defined as possessing at least one copy of the CBM 22, GH 10, and CBM 9 in the same orientation within a single polypeptide) in the GenBankTM database derived from other bacteria (supplemental Fig. 3). Interestingly, all of the organisms that possess a homolog of Xyn10A are thermophilic or hyperthermophilic bacteria, suggesting that this modular organization imparts an advantage to degrading xylan at elevated temperatures. Studies with XynA from Thermoanaerobacterium saccharolyticum (TsXynA), XynA from Thermotoga maritima (TmXynA), and XynC from Paenibacillus barcinonensis (PbXynC) found the N-terminal CBM 22 to be critical for imparting thermostability and thermophilicity to the respective enzymes (
      • Blanco A.
      • Díaz P.
      • Zueco J.
      • Parascandola P.
      • Javier Pastor F.I.
      A multidomain xylanase from a Bacillus sp. with a region homologous to thermostabilizing domains of thermophilic enzymes.
      ,
      • Lee Y.E.
      • Lowe S.E.
      • Henrissat B.
      • Zeikus J.G.
      Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.
      ,
      • Meissner K.
      • Wassenberg D.
      • Liebl W.
      The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed linkage β-1,3/β-1, 4-glucan.
      ). In addition to their thermostabilizing properties, the CBM 22 modules also possess carbohydrate binding activity. A polypeptide composed of both N-terminal CBM 22s of TmXynA bound both soluble xylan as well as mixed linkage β-1,3/β-1,4-glucan but did not bind to crystalline cellulose (
      • Meissner K.
      • Wassenberg D.
      • Liebl W.
      The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed linkage β-1,3/β-1, 4-glucan.
      ). Furthermore, a polypeptide composed only of the second CBM 22 of TmXynA possessed similar binding characteristics to the polypeptide containing both modules (
      • Meissner K.
      • Wassenberg D.
      • Liebl W.
      The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed linkage β-1,3/β-1, 4-glucan.
      ).
      The C-terminal CBM 9 of TmXynA and the noncellulosomal protein XynX from Clostridium thermocellum (CtXynX) possess cellulose binding activities and allow the respective enzymes to bind crystalline cellulose (
      • Selvaraj T.
      • Kim S.K.
      • Kim Y.H.
      • Jeong Y.S.
      • Kim Y.J.
      • Phuong N.D.
      • Jung K.H.
      • Kim J.
      • Yun H.D.
      • Kim H.
      The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding.
      ,
      • Winterhalter C.
      • Heinrich P.
      • Candussio A.
      • Wich G.
      • Liebl W.
      Identification of a novel cellulose-binding domain within the multidomain 120-kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima.
      ). Despite binding to crystalline cellulose, neither of these enzymes have the capacity to degrade this polysaccharide. Although the xylan-binding CBM 22 of these proteins likely aids in juxtaposing substrate and catalyst, the role of the cellulose-binding CBM 9 is less clear. However, in intact plant cell walls, xylans are found in close proximity to cellulose fibers; therefore, one possible function of the CBM 9 could be to aid in the separation and degradation of insoluble xylan fragments closely associated with cellulose fibrils. Although the precise function of CBM 9 in xylan degradation by these organisms remains unclear, the high level of conservation of this modular architecture in these proteins across diverse bacteria clearly suggests that these modules are integral to xylan degradation.
      Immunogold labeling and electron microscopy revealed that TmXynA is tethered to the outer membrane (toga) of T. maritima by a hydrophobic stretch of amino acids within the N-terminal signal peptide (
      • Liebl W.
      • Winterhalter C.
      • Baumeister W.
      • Armbrecht M.
      • Valdez M.
      Xylanase attachment to the cell wall of the hyperthermophilic bacterium Thermotoga maritima.
      ). Although there is a signal peptide within CpXyn10A, there is no significant homology between the signal peptides for the two xylanases nor is there a predicted signal peptidase II cleavage site that might facilitate transfer to a lipid moiety. Rather, CpXyn10A is most likely tethered to the surface of the bacterium via the three SLH repeats at the C terminus of the protein. SLH modules recognize and bind to pyruvylated cell wall polysaccharides (
      • Mesnage S.
      • Fontaine T.
      • Mignot T.
      • Delepierre M.
      • Mock M.
      • Fouet A.
      Bacterial SLH domain proteins are noncovalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation.
      ). The enzyme that mediates this modification is encoded by the csaB gene (
      • Mesnage S.
      • Fontaine T.
      • Mignot T.
      • Delepierre M.
      • Mock M.
      • Fouet A.
      Bacterial SLH domain proteins are noncovalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation.
      ), and a homolog of this gene is present within the genome of C. polysaccharolyticus (data not shown), indicating that this mechanism is active in this organism.
      Xyl3A is a bifunctional β-xylosidase/β-glucosidase with higher activity with pNPG relative to pNPX; however, the catalytic efficiency for xylo-oligosaccharides was several orders of magnitude higher than for cello-oligosaccharides indicating that cleaving debranched xylan fragments is the most likely activity of this protein. In addition to possessing the N-terminal (α/β)8 and C-terminal β-sandwich domains characteristic of GH 3 enzymes (
      • Varghese J.N.
      • Hrmova M.
      • Fincher G.B.
      Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase.
      ), Xyl3A also has a C-terminal fibronectin repeat 3-like (FnIII-like) domain (supplemental Fig. S1). FnIII domains are thought to have originated in animals and transferred to bacteria (
      • Bork P.
      • Doolittle R.F.
      Proposed acquisition of an animal protein domain by bacteria.
      ), where they exist almost exclusively in association with glycoside hydrolase enzymes (
      • Little E.
      • Bork P.
      • Doolittle R.F.
      Tracing the spread of fibronectin type III domains in bacterial glycohydrolases.
      ). The recent crystal structure of a three-domain GH 3 β-glucosidase from Thermotoga neapolitana (TnBgl3B) showed that the domain did not make contacts with the active site and adopts a FnIII fold that is distinct from those found in animals as well as those found in other bacterial glycoside hydrolases (
      • Pozzo T.
      • Pasten J.L.
      • Karlsson E.N.
      • Logan D.T.
      Structural and functional analyses of β-glucosidase 3B from Thermotoga neapolitana. A thermostable three-domain representative of glycoside hydrolase 3.
      ). Domain III of TnBgl3B was annotated as FnIII-like domain, and according to the Pfam database, 3290 of the total 3379 FnIII-like domains are associated with a GH 3 domain. Furthermore, 2949 of the total 6490 GH3 N domains are associated with FnIII-like domains, and the domain organization seen for Xyl3A is the most prevalent for GH 3 proteins in the Pfam database (data not shown). Despite the high abundance of proteins with this domain architecture, the role of this domain in GH3 enzymes is still unknown.
      XBP1 is the solute-binding component of an ABC transporter and specifically binds to xylo-oligosaccharides with a preference for xylotriose. Although four sequenced Thermoanaerobacterium spp. have uncharacterized homologs of XBP1 ranging in amino acid identity from 74 to 87%, the most closely related protein with demonstrated activity is XynE from G. stearothermophilus (52% identity). Similar to XBP1, GsXynE binds preferentially to xylotriose, although it prefers xylotetraose over xylobiose, which is in contrast to XBP1 (
      • Shulami S.
      • Zaide G.
      • Zolotnitsky G.
      • Langut Y.
      • Feld G.
      • Sonenshein A.L.
      • Shoham Y.
      A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.
      ). The gene encoding GsXynE is located within a 39.7-kb gene cluster containing hemicellulose utilization genes (
      • Shulami S.
      • Gat O.
      • Sonenshein A.L.
      • Shoham Y.
      The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.
      ), and its expression is regulated by a nearby two-component system (
      • Shulami S.
      • Zaide G.
      • Zolotnitsky G.
      • Langut Y.
      • Feld G.
      • Sonenshein A.L.
      • Shoham Y.
      A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.
      ). This arrangement is similar to that for XBP1 in that a putative two-component system lies just upstream of the ABC transporter genes, and it is likely that this system mediates the transcriptional response of these genes in the presence of xylan.
      Crystallographic studies of solute-binding proteins illustrate that specificity is mediated by interactions that are largely local to the binding pocket. A comparison of the co-crystal structure of the XBP1-xylotriose complex with other solute-binding proteins illustrates that the binding site for the polysaccharide ligand in XBP1 is optimized for xylotriose. For example, in the glucose-binding protein from T. thermophilus (PDB code 2B3B), the binding site is optimized for mono- and disaccharides, and longer oligosaccharides are occluded by the protrusion of a single residue (His-348) at site 3 and a depression of the loop equivalent to Thr-55 through Lys-60 at site 1. The structure of XBP1 illustrates a binding pocket with a contour that is optimized for trisaccharides, which contains polar residues that are suited for interactions with xylose residues at each of the three sites. Our structural and biochemical data further refine the idea that for solute-binding proteins ligand specificity is achieved within the confines of a highly conserved scaffold through modest changes at the binding site.
      The gene cluster identified in C. polysaccharolyticus appears to be targeted toward the utilization of xylans and specifically 4-O-methylglucuronoxylans as suggested by the presence of an α-glucuronidase gene within the cluster. The biological relevance of this gene cluster to xylan utilization by C. polysaccharolyticus is demonstrated by Q-PCR experiments that reveal many of these genes to be induced during growth on BWX relative to glucose. The genes located within this cluster encode the entire repertoire of enzymes required for the following: (a) transport xylan fragments across the cell membrane; (b) cleave branched oligosaccharides to monosaccharides; (c) metabolize xylose through the pentose-phosphate pathway, and (d) coordinate expression of xylanolytic genes in response to environmental availability of this substrate (Fig. 8).
      Figure thumbnail gr8
      FIGURE 8Schematic of a proposed pathway for metabolism of 4-O-MeGlcA-xylooligsaccharides in C. polysaccharolyticus. Xyn10A cleaves glucuronoxylan, liberating 2-O-α-4-O-methyl-α-d-glucuronosyl (4-O-MeGlcA) xylooligsaccharides or xylo-oligosaccharides, which are bound by XBP1 and transported into the cell via the ABC transporter. Inside the cell, the 4-O-MeGlcA-xylooligsaccharides are cleaved by Agu67A to yield xylooligsaccharides and 4-O-methyl-d-glucuronic acid. The xylo-oligosaccharides are then converted to xylose by the intracellularly located Xyl3A. In the xylose metabolism pathway, xylose isomerase converts xylose to xylulose, which is then converted to xylulose-5-P by xylulose kinase. Transketolase then catalyzes the rearrangement of xylulose-5-P and ribose-5-P to sedoheptulose-7-P and glyceraldehyde-3-P, and the transaldolase converts the two products to erythrose-4-P and fructose-6-P.
      The use of thermophilic organisms capable of fermenting both glucose and xylose in the production of biofuels is highly desirable in this emerging industry. In some instances, attempts are made to engineer an organism that can already use glucose to also metabolize xylan or xylose. The process often involves assembly of individual genes on a cassette to transform into the desired organism. The presence of the genes encoding the hydrolytic enzymes and the key enzymes of the pentose-phosphate pathway in a cluster makes it easier to transfer a co-evolved molecular machinery to confer the xylanolytic phenotype.

      Acknowledgments

      We thank Young-Hwan Moon for technical assistance.

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