Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.     

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana

The synthesis, modification, and breakdown of carbohydrates is one of the most fundamentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1-2% of an organism's genes to glycoside hydrolases and glycosyltransferases alone. In plants, these processes are of particular importance for cell-wall synthesis and expansion. starch metabolism, defence against pathogens, symbiosis and signalling. Here we present an analysis of over 730 open reading frames representing the two main classes of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, in the genome of Arabidopsis thaliana. The vast importance of these enzymes in cell-wall formation and degradation is revealed along with the unexpected dominance of pectin degradation in Arabidopsis, with at least 170 open-reading frames dedicated solely to this task.     

Microbial carbohydrate esterases deacetylating plant polysaccharides

Several plant polysaccharides are partially esterified with acetic acid. One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However, this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families, specifically in CE families 1–7 and 16, originally assigned as acetylxylan esterases, the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-β-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan, but they have not been classified yet. The enzymes present in CE families 1–7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates, i.e. esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception, the enzymes of all mentioned CE families belong to serine type esterases. CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families, however, there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates.     

Hierarchical classification of glycoside hydrolases

This review deals with structural and functional features of glycoside hydrolases, a widespread group of enzymes present in almost all living organisms. Their catalytic domains are grouped into 120 amino acid sequence-based families in the international classification of the carbohydrate-active enzymes (CAZy database). At a higher hierarchical level some of these families are combined in 14 clans. Enzymes of the same clan have common evolutionary origin of their genes and share the most important functional characteristics such as composition of the active center, anomeric configuration of cleaved glycosidic bonds, and molecular mechanism of the catalyzed reaction (either inverting, or retaining). There are now extensive data in the literature concerning the relationship between glycoside hydrolase families belonging to different clans and/or included in none of them, as well as information on phylogenetic protein relationship within particular families. Summarizing these data allows us to propose a multilevel hierarchical classification of glycoside hydrolases and their homologs. It is shown that almost the whole variety of the enzyme catalytic domains can be brought into six main folds, large groups of proteins having the same three-dimensional structure and the supposed common evolutionary origin.     

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.     

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.     

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.     

Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis.

Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

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.     

Different techniques reveal the difference of community structure and function of fungi from root and rhizosphere of Salvia miltiorrhiza Bunge

• Fungi have essential functions in plant health and performance. However, the plant-associated functions of many cultured fungi have not been established in detail.
• Here, the fungal species diversity in Salvia miltiorrhiza roots and rhizosphere was assessed for the first time using culturomics and high-throughput sequencing. We present a comprehensive functional metagenomic analysis of these fungi and verified activity of cellulase and chitinase predicted in the metagenomic analysis.
• We first collected and cultured fungi from the root and rhizosphere of S. miltiorrhiza. We found 92 species across 37 families and five phyla, with Ascomycota being dominant. Many rDNA internal transcribed spacer sequences could not be assigned to lower taxonomic levels. There were 19 genera of endophytic fungi and 37 genera of rhizosphere fungi. The culturomics approach had lower taxonomic diversity than high-throughput sequencing, but some fungi were only found in cultures. Structural analyses indicated that the dominant species differed in cultured and non-cultured samples at other levels, apart from the phylum level. Functional analysis mapped 223 carbohydrate enzyme families and 393 pathways in the CAZy and KEGG databases, respectively. The most abundant families were glycoside hydrolases and those involved in carbohydrate metabolism. As predicted by metagenomics, we experimentally verified cellulase and chitinase activity for 29 and 74 fungi, respectively.
• We provide the first evidence of biomass recycling by fungi that are associated with plants. Culturing is essential to reveal the hidden microbial community and critical functions in plant–microbe interactions.

     

Systemic use of “limping” enzymes in plant cell walls

Diversity of proteins and enzymes engaged in carbohydrate metabolism is vast. This is related to the fact that plants contain the greater part of biospheric carbohydrates, whose structures are extremely diverse as well. In plant genomes, proteins involved in carbohydrate metabolism are grouped into numerous families, and each of them may include tens of sequences. This is especially typical of enzymes modifying polysaccharides of the cell wall. Expression of genes encoding such proteins is finely tuned. It may differ in different tissues and organs and depends on stages of development of the entire plant and its particular cells. However, certain genes, including highly expressive ones, encode enzymes with “limping” catalytic centers, which may be unable to conduct reactions characteristic of the particular enzymatic family. The review surveys examples of such proteins and discusses causes of their origin and possible functions.     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Insights into plant cell wall degradation from the genome sequence of the soil bacterium Cellvibrio japonicus

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 2-40(T). 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.     

Furanosidase superfamily: Search of homologues

The furanosidase superfamily contains the GH32, GH43, GH62, GH68, GH117, DUF377 (GH130), and DUF1861 families of glycoside hydrolases and their homologues. Catalytic domains of these families have five-bladed β-propeller tertiary structure. Iterative screening of the protein database supports of their relationship as well as evolutionary connections with domains from GH33 and GH93 families of glycoside hydrolases. The latter two have the structure of the six-bladed β-propeller. Among detected homologues we found 441 unclassified proteins. These proteins are combined into 39 groups based on homology: FURAN1-FURAN39. FURAN8 and FURAN36 can be considered as separate subfamilies within the GH43 and GH32 families of glycoside hydrolases, respectively. The remaining 37 groups are new families of hypothetical glycoside hydrolases.     

Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum

Numerous bacterial and fungal organisms have evolved elaborate sets of modular glycoside hydrolases and similar enzymes aimed at the degradation of polymeric carbohydrates. Presently, on the basis of sequence similarity catalytic modules of these enzymes have been classified into 90 families. Representatives of a particular family display similar fold and catalytic mechanisms. However, within families distinctions occur with regard to enzymatic properties and type of activity against carbohydrate chains. Cellobiohydrolase CbhA from Clostridium thermocellum is a large seven-modular enzyme with a catalytic module belonging to family 9. In contrast to other representatives of that family possessing only endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an exocellulase. The crystal structures of the combination of the immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT) are reported here. The detailed analysis of these structures reveals that, while key catalytic residues and overall fold are conserved in this enzyme and those of other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off after the -2 subsite. This feature which is created by an extension and altered conformation of a single loop region explains the inability of the active site of CbhA to accommodate a long cellulose chain and to cut it internally. This altered loop region is responsible for the exocellulolytic activity of the enzyme.     

Investigating the Function of an Arabinan Utilization Locus Isolated from a Termite Gut Community

Biocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genus Bacteroides and encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar. In this respect and considering the phenotype of the metagenomic clone, it was noted that among the ORFs, there are four putative arabinose-specific glycoside hydrolases, two from family GH43 and two from GH51. In this study, a thorough bioinformatics analysis of the metagenomic clone gene cluster has been performed and the four aforementioned glycoside hydrolases have been characterized. Together, the results provide evidence that the gene cluster is a polysaccharide utilization locus dedicated to the breakdown of the arabinan component in pectin and related substrates. Characterization of the two GH43 and the two GH51 glycoside hydrolases has revealed that each of these enzymes displays specific catalytic capabilities and that when these are combined the enzymes act synergistically, increasing the efficiency of arabinan degradation.     

Structural enzymology of carbohydrate-active enzymes: implications for the post-genomic era

Simple and complex carbohydrates have been described as "the last frontier of molecular and cell biology". The enzymes that are required for the synthesis and degradation of these compounds provide an enormous challenge in the post-genomic era. This reflects both the extreme chemical and functional diversity of sugars and the difficulties in characterizing both the substrates and the enzymes themselves. The vast myriad of enzymes involved in the synthesis, modification and degradation of oligosaccharides and polysaccharides is only just being unveiled by genomic sequencing. These so-called "carbohydrate-active enzymes" lend themselves to classification by sensitive sequence similarity detection methods. The modularity, often extremely complex, of these enzymes must first be dissected and annotated before high throughput characterization or "structural genomics" approaches may be employed. Once achieved, modular analysis also permits collation of a detailed "census" of carbohydrate-active enzymes for a whole organism or throughout an ecosystem. At the structural level, improvements in X-ray crystallography have opened up a three-dimensional understanding of the way these enzymes work. The mechanisms of many of the glycoside hydrolase families are becoming clearer, yet glycosyltransferases are only slowly revealing their secrets. What is clear from the genomic and structural data is that if we are to harness the latent power of glycogenomics, scientists must consider distant sequence relatives revealed by the sequence families or other sensitive detection methods.