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1.  Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont 
Nature chemical biology  2009;6(2):125-132.
Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are α-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca2+-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca2+ plays a key catalytic role in helping distort the mannoside away from its ground-state 4C1 chair conformation toward the transition state.
doi:10.1038/nchembio.278
PMCID: PMC3942423  PMID: 20081828
2.  Evidence That GH115 α-Glucuronidase Activity, Which Is Required to Degrade Plant Biomass, Is Dependent on Conformational Flexibility* 
Background: The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown.
Results: Bacteroides ovatus GH115 glucuronidase is a dimeric enzyme that contains a flexible active site pocket.
Conclusion: The assembly of the catalytic apparatus of the glucuronidase requires substantial conformational changes.
Significance: Conformational changes are highly unusual in glycoside hydrolases.
The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.
doi:10.1074/jbc.M113.525295
PMCID: PMC3879575  PMID: 24214982
Bioenergy; Enzyme Structure; Glycoside Hydrolases; Plant Cell Wall; X-ray Crystallography
3.  Influence of a Mannan Binding Family 32 Carbohydrate Binding Module on the Activity of the Appended Mannanase 
Applied and Environmental Microbiology  2012;78(14):4781-4787.
In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.
doi:10.1128/AEM.07457-11
PMCID: PMC3416382  PMID: 22562994
4.  Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan* 
The Journal of Biological Chemistry  2012;288(7):4799-4809.
Background: Carbohydrate binding modules (CBMs) contribute to the enzymatic degradation of complex polysaccharide structures.
Results: New CBMs display specificity for decorated glucans through an extensive hydrophobic platform that interacts with both backbone and side chain structures.
Conclusion: CBMs that bind to complex β-glucans exploit different components of these ligands as specificity determinants.
Significance: CBMs can utilize the side chains of decorated glucans as specificity determinants.
Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.
doi:10.1074/jbc.M112.432781
PMCID: PMC3576085  PMID: 23229556
Carbohydrate-binding Protein; Isothermal Titration Calorimetry; Plant Cell Wall; Protein Structure; X-ray Crystallography
5.  Recognition and Degradation of Plant Cell Wall Polysaccharides by Two Human Gut Symbionts 
PLoS Biology  2011;9(12):e1001221.
Competition for nutrients contained in diverse types of plant cell wall-associated polysaccharides may explain the evolution of substrate-specific catabolic gene modules in common bacterial members of the human gut microbiota.
Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs) that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target unique suites of available polysaccharides, a theme that likely applies to disparate bacteria from the gut and other habitats.
Author Summary
Bacteria inhabiting the human gut are critical for digestion of the plant-derived glycans that compose dietary fiber. Enzymes produced by the human body cannot degrade these abundant dietary components, and without bacterial assistance they would go unused. We investigated the molecular strategies employed by two species belonging to one of the most abundant bacterial groups in the human colon (the Bacteroidetes). Our results show that each species has evolved to degrade a unique subset of glycans; this specialization is reflected in their respective genomes, each of which contains numerous separate gene clusters involved in metabolizing plant fiber polysaccharides or glycans present in secreted mucus. Each glycan-specific gene cluster produces a related series of membrane-associated proteins which together serve to bind and degrade a specific glycan. Expression of each glycan-specific gene cluster is controlled by an environmental sensor that responds to the presence of a unique molecular signature contained in the substrate that it targets. These results provide a view of how related bacterial species have diverged into different carbohydrate niches by evolving genes that sense and degrade unique suites of available polysaccharides, a process that likely applies to disparate bacteria from the gut and other habitats.
doi:10.1371/journal.pbio.1001221
PMCID: PMC3243724  PMID: 22205877
6.  Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725 
Nucleic Acids Research  2011;39(8):3240-3254.
Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼80°C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.
doi:10.1093/nar/gkq1281
PMCID: PMC3082886  PMID: 21227922
7.  Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase 
PLoS ONE  2010;5(2):e9006.
Background
The enzymatic hydrolysis of α−mannosides is catalyzed by glycoside hydrolases (GH), termed α−mannosidases. These enzymes are found in different GH sequence–based families. Considerable research has probed the role of higher eukaryotic “GH38” α−mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 α−mannosidase II, which has been shown to be a retaining α−mannosidase that targets both α−1,3 and α−1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)5(GlcNAc)2 hybrid N-glycans to GlcNAc(Man)3(GlcNAc)2. Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 α−mannosidases whose activity and specificity is unknown.
Methodology/Principal Findings
Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an α−mannosidase with specificity for α−1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 Å resolution and in complex with the inhibitor swainsonine (Ki 18 µM) at 2.6 Å, reveals a canonical GH38 five-domain structure in which the catalytic “–1” subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn2+ ion. In contrast, the “leaving group” subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity.
Conclusions/Significance
Although the in vivo function of this streptococcal GH38 α−mannosidase remains unknown, it is shown to be an α−mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.
doi:10.1371/journal.pone.0009006
PMCID: PMC2815779  PMID: 20140249
8.  Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus▿ †  
Journal of Bacteriology  2008;190(15):5455-5463.
The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40T. Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.
doi:10.1128/JB.01701-07
PMCID: PMC2493263  PMID: 18556790
9.  CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose 
Journal of Bacteriology  2003;185(2):391-398.
The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.
doi:10.1128/JB.185.2.391-398.2003
PMCID: PMC145334  PMID: 12511483
10.  The Membrane-Bound α-Glucuronidase from Pseudomonas cellulosa Hydrolyzes 4-O-Methyl-d-Glucuronoxylooligosaccharides but Not 4-O-Methyl-d-Glucuronoxylan 
Journal of Bacteriology  2002;184(17):4925-4929.
The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-d-glucuronoxylans are extensively decorated with 4-O-methyl-d-glucuronic acid, which is cleaved from the polysaccharides by α-glucuronidases. In this report we describe the primary structures of the α-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the α-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with α-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad α-glucuronidase released 4-O-methyl-d-glucuronic acid from 4-O-methyl-d-glucuronoxylooligosaccharides but not from 4-O-methyl-d-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-d-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.
doi:10.1128/JB.184.17.4925-4929.2002
PMCID: PMC135289  PMID: 12169619
11.  Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11 
Journal of Bacteriology  2002;184(15):4124-4133.
Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.
doi:10.1128/JB.184.15.4124-4133.2002
PMCID: PMC135193  PMID: 12107129
12.  Control of Synthesis of Functional mRNA Coding for Phenylalanine Ammonia-Lyase from Rhodosporidium toruloides 
Journal of Bacteriology  1983;153(3):1147-1154.
The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.
Images
PMCID: PMC221757  PMID: 6826518
13.  The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions 
PLoS Biology  2009;7(3):e1000071.
Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
Author Summary
Proteins that display multiple activities have typically evolved through the recruitment of different domains, each of which has a specific function. Thus, in a multifunctional protein, the different activities are mediated by spatially distinct domains such that a single domain can provide the specific chemical requirements for one activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within a single-domain enzyme is likely to be a rare event, as it would compromise the existing activity of the protein when a new function evolves. Nonetheless, a potential example of multifunctional recruitment into a single protein domain is provided by an enzyme that contains a cellulase enzyme module and a discrete noncatalytic cellulose-binding module. In this article, we show that the cellulose-binding module displays esterase activity and that these diverse activities are housed within the same site on the protein. Structural analysis of the enzyme reveals that its catalytic residues also contribute to the noncatalytic cellulose-binding function. This report provides a rare example of “gene sharing,” whereby the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the catalyst.
The active of site of an esterase enzyme has acquired a noncatalytic carbohydrate-binding function without compromising its catalytic activity, providing support for the "gene sharing" model of protein diversification.
doi:10.1371/journal.pbio.1000071
PMCID: PMC2661963  PMID: 19338387
14.  Complete Genome Sequence of the Complex Carbohydrate-Degrading Marine Bacterium, Saccharophagus degradans Strain 2-40T 
PLoS Genetics  2008;4(5):e1000087.
The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.
Author Summary
A segment of the global marine carbon cycle that has been poorly characterized is the mineralization of complex polysaccharides to carbon dioxide, a greenhouse gas. It also remained a mystery whether prokaryotes mineralize plant/algal cell walls and woody material in the oceans via carbohydrase systems and, if so, which organisms are involved. We have analyzed the complete genome sequence of the marine bacterium Saccharophagus degradans to better ascertain the potential role of prokaryotes in marine carbon transformation. We discovered that S. degradans, which is related to a number of other newly discovered marine strains, has an unprecedented quantity and diversity of carbohydrases, including the first characterized marine cellulose system. In fact, extensive analysis of the S. degradans genome sequence and functional followup experiments identified an extensive collection of complete enzyme systems that degrade more than 10 complex polysaccharides. These include agar, alginate, and chitin, altogether representing an extraordinary range of catabolic capability. Genomic analyses further demonstrated that the carbohydrases are unusually modular; sequence comparisons revealed that many of the functional modules were acquired by lateral transfer. These results suggest that the prokaryotic contribution to marine carbon fluxes is substantial and cannot be ignored in predictions of climate change.
doi:10.1371/journal.pgen.1000087
PMCID: PMC2386152  PMID: 18516288

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