The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

Complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T

Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S. degradans genome has been sequenced to completion, and more than 180 open reading frames have been identified that encode carbohydrases. Over half of these are likely to act on plant cell wall polymers. In fact, there appears to be a full array of enzymes that degrade and metabolize plant cell walls. Genomic and proteomic analyses reveal 13 cellulose depolymerases complemented by seven accessory enzymes, including two cellodextrinases, three cellobiases, a cellodextrin phosphorylase, and a cellobiose phosphorylase. Most of these enzymes exhibit modular architecture, and some contain novel combinations of catalytic and/or substrate binding modules. This is exemplified by endoglucanase Cel5A, which has three internal family 6 carbohydrate binding modules (CBM6) and two catalytic modules from family five of glycosyl hydrolases (GH5) and by Cel6A, a nonreducing-end cellobiohydrolase from family GH6 with tandem CBM2s. This is the first report of a complete and functional cellulase system in a marine bacterium with a sequenced genome.     

Plant glycoside hydrolases involved in cell wall polysaccharide degradation.

The cell wall plays a key role in controlling the size and shape of the plant cell during plant development and in the interactions of the plant with its environment. The cell wall structure is complex and contains various components such as polysaccharides, lignin and proteins whose composition and concentration change during plant development and growth. Many studies have revealed changes in cell walls which occur during cell division, expansion, and differentiation and in response to environmental stresses; i.e. pathogens or mechanical stress. Although many proteins and enzymes are necessary for the control of cell wall organization, little information is available concerning them. An important advance was made recently concerning cell wall organization as plant enzymes that belong to the superfamily of glycoside hydrolases and transglycosidases were identified and characterized; these enzymes are involved in the degradation of cell wall polysaccharides. Glycoside hydrolases have been characterized using molecular, genetic and biochemical approaches. Many genes encoding these enzymes have been identified and functional analysis of some of them has been performed. This review summarizes our current knowledge about plant glycoside hydrolases that participate in the degradation and reorganisation of cell wall polysaccharides in plants focussing particularly on those from Arabidopsis thaliana.     

Enzymatic Deconstruction of Backbone Structures of the Ramified Regions in Pectins

The pectic enzymes are a diverse group of enzymes that collectively degrade pectin, a mixture of highly heterogeneous and branched polysaccharides rich in d-galacturonic acids forming a major component of the primary cell wall of plants. This review covers key enzymes that function to deconstruct the “ramified region” of pectin. The enzymes include glycoside hydrolases and polysaccharide lyases that degrade complex pectic domains consisting of rhamnogalacturonans, xylogalacturonans, and other heterogeneous polymers. The chemical nature of the pectic substrates for the enzymes is presented. The biochemical properties of the enzymes, the mechanisms of enzyme actions, and related structures and functions, are described. Applications of these enzymes in fruit juice processing and in the production of bioactive compounds, as well as their technological relevance to the deconstruction of cell wall structures for biomass conversion are discussed.     

X4 modules represent a new family of carbohydrate-binding modules that display novel properties

The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.     

The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T

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.     

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.     

Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum

The digestion of the plant cell wall requires the concerted action of a diverse repertoire of enzyme activities. An important component of these hydrolase consortia are arabinofuranosidases, which release L-arabinofuranose moieties from a range of plant structural polysaccharides. The anaerobic bacterium Clostridium thermocellum, a highly efficient plant cell wall degrader, possesses a single alpha-L-arabinofuranosidase (EC 3.2.1.55), CtAraf51A, located in GH51 (glycoside hydrolase family 51). The crystal structure of the enzyme has been solved in native form and in 'Michaelis' complexes with both alpha-1,5-linked arabinotriose and alpha-1,3 arabinoxylobiose, both forming a hexamer in the asymmetric unit. Kinetic studies reveal that CtAraf51A, in contrast with well-characterized GH51 enzymes including the Cellvibrio japonicus enzyme [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614], catalyses the hydrolysis of alpha-1,5-linked arabino-oligosaccharides and the alpha-1,3 arabinosyl side chain decorations of xylan with equal efficiency. The paucity of direct hydrogen bonds with the aglycone moiety and the flexible conformation adopted by Trp(178), which stacks against the sugar at the +1 subsite, provide a structural explanation for the plasticity in substrate specificity displayed by the clostridial arabinofuranosidase.     

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.     

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.     

Functional analyses of multiple lichenin-degrading enzymes from the rumen bacterium Ruminococcus albus 8

Ruminococcus albus 8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence of R. albus revealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived from R. albus, to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.     

Importance of hydrophobic and polar residues in ligand binding in the family 15 carbohydrate-binding module from Cellvibrio japonicus Xyn10C

Modular glycoside hydrolases that degrade the plant cell wall often contain noncatalytic carbohydrate-binding modules (CBMs) that interact with specific polysaccharides within this complex macromolecule. CBMs, by bringing the appended catalytic module into intimate and prolonged association with the substrate, increase the rate at which these enzymes are able to hydrolyze glycosidic bonds. Recently, the crystal structure of the family 15 CBM (CBM15) from Cellvibrio japonicus (formerly Pseudomonas cellulosa) Xyn10C was determined in complex with the ligand xylopentaose. In this report we have used a rational design approach, informed by the crystal structure of the CBM15-ligand complex, to probe the importance of hydrophobic stacking interactions and both direct and water-mediated hydrogen bonds in the binding of this protein to xylan and xylohexaose. The data show that replacing either Trp 171 or Trp 186, which stack against xylose residues n and n + 2 in xylopentaose, with alanine abolished ligand binding. Similarly, replacing Asn 106, Gln 171, and Gln 217, which make direct hydrogen bonds with xylopentaose, with alanine greatly reduced the affinity of the protein for its saccharide ligands. By contrast, disrupting water-mediated hydrogen bonds between CBM15 and xylopentaose by introducing the mutations S108A, Q167A, Q221A, and K223A had little effect on the affinity of the protein for xylan or xylohexaose. These data indicate that CBM15 binds xylan and xylooligosaccharides via the same interactions and provide clear evidence that direct hydrogen bonds are a key determinant of affinity in a type B CBM. The generic importance of these data is discussed.     

Biochemical analyses of multiple endoxylanases from the rumen bacterium Ruminococcus albus 8 and their synergistic activities with accessory hemicellulose-degrading enzymes

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R. albus 8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of R. albus 8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a β-xylosidase, an α-l-arabinofuranosidase, and an α-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by R. albus 8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.     

Fungal lysis by a soil bacterium fermenting cellulose

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.     

The distribution of polysaccharide-degrading enzymes in the bovine rumen digesta ecosystem

The location and level of activity of the principal polysaccharidases and glycoside hydrolases involved in the degradation of plant structural and storage polysaccharides were monitored in microbial populations isolated from liquid and particulate phases of bovine rumen digesta. The three principal subpopulations, and their constituent subgroups studied, all contained polysaccharide depolymerizing enzymes; however, the specific activities of the enzymes that degraded the plant cell wall structural polymers were highest within the adherent particle-associated populations. Separate functional groups of organisms could be recongnized in the particle-associated population by their distinctive enzyme profiles.     

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.