Structure- and context-based analysis of the GxGYxYP family reveals a new putative class of glycoside hydrolase.

BackgroundGut microbiome metagenomics has revealed many protein families and domains found largely or exclusively in that environment. Proteins containing the GxGYxYP domain are over-represented in the gut microbiota, and are found in Polysaccharide Utilization Loci in the gut symbiont Bacteroides thetaiotaomicron, suggesting their involvement in polysaccharide metabolism, but little else is known of the function of this domain.ResultsGenomic context and domain architecture analyses support a role for the GxGYxYP domain in carbohydrate metabolism. Sparse occurrences in eukaryotes are the result of lateral gene transfer. The structure of the GxGYxYP domain-containing protein encoded by the BT2193 locus reveals two structural domains, the first composed of three divergent repeats with no recognisable homology to previously solved structures, the second a more familiar seven-stranded β/α barrel. Structure-based analyses including conservation mapping localise a presumed functional site to a cleft between the two domains of BT2193. Matching to a catalytic site template from a GH9 cellulase and other analyses point to a putative catalytic triad composed of Glu272, Asp331 and Asp333.ConclusionsWe suggest that GxGYxYP-containing proteins constitute a novel glycoside hydrolase family of as yet unknown specificity

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies

Transglycosylation by Glycoside Hydrolases - Production and modification of alkyl glycosides

To enable the transition to a green, bio-based economy, an extensive enzymatic toolbox competitive to traditional chemical procedures is needed. One strong area for enzymes is carbohydrate chemistry, due to the over-functionalized nature of carbohydrates, difficult to handle in traditional chemistry. Glycosylation can be catalyzed by four main classes of enzymes, glycosyltransferases, glycoside phosphorylases, transglycosylases and glycoside hydrolases. For industrial implementation, transglycosylases are ideal catalysts that do not need the expensive activated donors associated with glycoside phosphorylases and glycosyltransferases. In addition, they completely lack the hydrolytic activity intrinsic in the closely related glycoside hydrolases. Unfortunately, very few transglycosylases with limited substrate specificities exist in nature, while a wide abundance of glycoside hydrolases are available. To expand the enzymatic toolbox for synthetic chemists it would be favorable to convert glycoside hydrolases into transglycosylases, by limiting their hydrolytic activity. This dissertation investigates the transglycosylation activity of glycoside hydrolases with synthesis and modification of alkyl glycosides, a widely applicable type of surfactants, as model reactions. Reduced hydrolysis for β-glycosidases from the thermophilic Thermotoga neapolitana was achieved through protein engineering, limiting water content and increasing pH. Complete elimination of the hydrolytic activity with maintained transglycosylation was achieved for synthesis of hexyl-β-D-glucoside and the factors resulting in the success are discussed. In addition, extension of the glycosidic part of alkyl glycosides through the coupling activity of cyclodextrin glucanotransferases is explored. An enzyme kinetics study was undertaken to deduce the optimal reaction conditions to promote coupling for a commercial enzyme. Moreover, a novel cyclodextrin glucanotransferase from Carboxydocella species was characterized, shown to have good coupling activity with γ-cyclodextrins as donor. This previously poorly studied donor can be used to extend the range of alkyl glycosides that can be produced and thereby the number of applications available

Enzymatic conversion of β-mannans: Analysing, evaluating and modifying transglycosylation properties of glycoside hydrolases

Retaining glycoside hydrolases are enzymes that catalyse the breakdown down of glycans through hydrolysis. Due to the double-replacement mechanism of the retaining glycoside hydrolases (GHs), which form an intermediate with part of the glycan covalently attached to the enzyme, some GHs are able to catalyse synthesis reactions called transglycosylation. In transglycosylation reactions a hydroxyl-containing molecule (acceptor), other than water, acts as a nucleophile which releases the glycan moiety from the covalent intermediate while forming a new glycoside (transglycosylation product). The transglycosylation reaction can be used to transform renewable starting materials such as plant hemicellulose to valuable products, which is discussed in the thesis. The work presented in the thesis have explored how GHs interact with glycans and how different aspects of transglycosylation reactions affect the final yield of transglycosylation products. The presented work explores how the open active site structure of two GH26 β-mannanases have made them well adapted to act on heavily galactosylated hemicellulosic β-mannan polysaccharides (Paper I and II). In addition Paper I and II explore how substitutions of amino acids in glycan interacting subsites can lead to changes in catalytic properties and how the two GH26 β-mannanases productively interacts with oligosaccharides. The work also examines how variants of GHs can have improved transglycosylation capacity compared to their wildtype counterparts (Paper III and V). It investigates how the elimination of saccharide interactions in the +2 subsites can lead to improved transglycosylation capacity in a variant of the GH5 β-mannanase TrMan5A (PaperIII). The TrMan5A variant displayed greatly improved transglycosylation capacity at the early timepoints. Observed secondary (product) hydrolysis at later times highlighted the importance of analysing prolonged reaction times to determine suitable reaction termination. Paper III also demonstrated how enzyme synergy can lead to increased transglycosylation yields, when TrMan5A and a guar α-galactosidase was used in co-incubations where a galactomannan was used as the glycosyl donor. α-Galactosidases were further studied in Paper IV, where thetransglycosylation capacity of two different α-galactosidases were explored with different glycosyl donors and acceptor molecules. The study showed that the guar α-galactosidase was able to utilise a wide variety of acceptor molecules and glycosyl donors, further expanding potential transglycosylation products that may be produced with the enzyme. Paper IV further highlights the negative effects secondary hydrolysis may have on transglycosylation yields. The presented work also shows how targeting highly conserved residues within a glycoside hydrolase family can be used to quickly generate GH variants with improved transglycosylation capacity compared to the wild type GH (Paper V). The method relies on protein sequence data and does not require structural knowledge of the target enzyme. Furthermore, the method generates few variants (evolution (100s to 1000s) while it appears to be generally applicable as it was successfully applied to six different GH families covering varying specificities. Improvements was, in part, indicated to be associated with reduced secondary hydrolysis in several of the six GH families in the study. The results presented in the thesis have expanded the knowledge of different factors that affects and can be manipulated in order to improve the transglycosylation capacity in retaining glycoside hydrolases. The work presented in the thesis will help further enzymatic synthesis approaches utilising renewable raw-materials

Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

Glycoside hydrolases (GH) catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/). This database integrates multiple data types including the structural features, orthologous relationships, mutant availability, and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification

GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

BACKGROUND: As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. RESULTS: The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20). CONCLUSION: The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α)(8)-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans

Transcriptome Profile of Trichoderma harzianum IOC-3844 Induced by Sugarcane Bagasse

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. in the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. the sequencing generated 14.7 Gbp for downstream analyses. de novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. the present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Campinas UNICAMP, CBMEG, Campinas, SP, BrazilBrazilian Ctr Res Energy & Mat CNPEM, Brazilian Bioethanol Sci & Technol Lab CTBE, Campinas, SP, BrazilUniv São Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Campinas UNICAMP, Dept Plant Biol, Inst Biol, Campinas, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilWeb of Scienc

Probing of Carbohydrate-Protein Interactions Using Galactonoamidine Inhibitors

Glycoside hydrolases are ubiquitous and one of the most catalytically proficient enzymes known, and thus understanding their mechanisms are crucial. Most research has focused on the interaction of the glycon of substrates and their inhibitors within the active site of glycoside hydrolases. The inhibitors employed to probe these interactions generally had small aglycons (i.e. a hydrogen atom, amidines, small aliphatic groups, or benzyl groups). Here, the interactions of the aglycon with glycoside hydrolases are examined by probing the active sites with a library of 25 galactonoamidines. The studies described in this dissertation aim to increase the understanding of stabilization of the transition state by glycoside hydrolases, which allows for the acceleration of substrate hydrolysis by the enzymes up to 1017 over non-enzymatic hydrolysis. To understand this stabilization, the active sites of beta-galactosidases from Aspergillus oryzae, bovine liver, and Escherichia coli were evaluated using spectroscopic, molecular docking, and modeling analyses to determine transition state analogs (TSAs) and how the TSAs interact within the active site of glycoside hydrolases. The probing with the galactonoamidine library revealed hydrophobic interactions, pi-pi interactions, and CH-pi interactions within the active sites to varying extent. Further, three TSAs were found for the hydrolysis of substrates by beta-galactosidase (A. oryzae), and two TSAs for the beta-galactosidases from bovine liver and E. coli. Upon TSA binding to the three beta-galactosidases, conformational changes occurred to stabilize the galactonoamidines within the active sites, which did not occur when fortuitous binders interacted with the enzyme. The conformational changes within the active sites of beta-galactosidases from bovine liver and E. coli closes off the active site via a loop movement resulting in a substantially higher binding affinity than those observed with beta-galactosidase (A. oryzae). A subsequent evaluation of galactonoamidine specificity in the presence of other proteins revealed an increase of inhibitory activity two orders of magnitude more than a purified beta-galactosidase (E. coli)

High resolution crystal structure of the Endo-N-acetyl-beta-D-glucosaminidase responsible for the deglycosylation of hypocrea jecorina cellulases

Endo-N-acetyl-beta-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-beta-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified. Here, the expression, purification and the 1.3 angstrom resolution structure of the ENGase ( Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) are reported. Although the mature protein is C-terminally processed with removal of a 46 amino acid peptide, the protein has a complete (beta/alpha) 8 TIM-barrel topology. In the active site, the proton donor (E131) and the residue stabilizing the transition state (D129) in the substrate assisted catalysis mechanism are found in almost identical positions as in the bacterial GH18 ENGases: Endo H, Endo F1, Endo F3, and Endo BT. However, the loops defining the substrate-binding cleft vary greatly from the previously known ENGase structures, and the structures also differ in some of the alpha-helices forming the barrel. This could reflect the variation in substrate specificity between the five enzymes. This is the first three-dimensional structure of a eukaryotic endo-N-acetyl-beta-D-glucosaminidase from glycoside hydrolase family 18. A glycosylation analysis of the cellulases secreted by a Hypocrea jecorina Endo T knock-out strain shows the in vivo function of the protein. A homology search and phylogenetic analysis show that the two known enzymes and their homologues form a large but separate cluster in subgroup B of the fungal chitinases. Therefore the future use of a uniform nomenclature is proposed