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Here the statistics concerning X-ray data processing and structure refinement are given, together with the substrate preference analysis for ThBgl1 and ThBgl2. Finally, the analysis of the influence of temperature and pH on the activities of both enzymes are shown.

Plant biomass is a low-cost and abundant source of carbohydrates for production of fuels, "green" chemicals and materials. Currently, biochemical conversion of the biomass into sugars via enzymatic hydrolysis is the most viable technology. Here, the role of carbohydrate binding modules (CBMs) in the disruption of insoluble polysaccharide structures and their capacity to enhance cellulase-promoted lignocellulosic biomass hydrolysis was investigated. We show that CBM addition promotes generation of additional reducing ends in the insoluble substrate by cellulases. On the contrary, bovine serum albumin (BSA), widely used in prevention of a non-specific protein binding, causes an increase in soluble reducing-end production, when applied jointly with cellulases. We demonstrate that binding of CBMs to cellulose is non-homogeneous, irreversible and leads to its amorphisation. Our results also reveal effects of CBM-promoted amorphogenesis on cellulose hydrolysis by cellulases.

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of β-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the “Newton’s cradle” catalytic mechanism recently proposed for this subfamily.

Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. β-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 β-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-β- d -glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-β- d -xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 A resolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/β sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.

β-glucosidases are glycoside hydrolases able to cleave small and soluble substrates, thus producing monosaccharides. These enzymes are distributed among families GH1, GH2, GH3, GH5, GH9, GH30 and GH116, with GH1 and GH3 being the most relevant families with characterized enzymes to date. A recent transcriptomic analysis of the fungus Trichoderma harzianum, known for its increased β-glucosidase activity as compared to Trichoderma reesei, revealed two enzymes from family GH1 with high expression levels. Here we report the cloning, recombinant expression, purification and crystallization of these enzymes, ThBgl1 and ThBgl2. A close inspection of the enzymatic activity of these enzymes surprisingly revealed a marked difference between them despite the sequence similarity (53%). ThBgl1 has an increased tendency to catalyze transglycosylation reaction while ThBgl2 acts more as a hydrolyzing enzyme. Detailed comparison of their crystal structures and the analysis of the molecular dynamics simulations reveal the presence of an asparagine residue N186 in ThBgl2, which is replaced by the phenylalanine F180 in ThBgl1. This single amino acid substitution seems to be sufficient to create a polar environment that culminates with an increased availability of water molecules in ThBgl2 as compared to ThBgl1, thus conferring stronger hydrolyzing character to the former enzyme.

Made available in DSpace on 2018-11-26T15:29:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme. Univ Sao Paulo, Sao Carlos Inst Phys, BR-13566590 Sao Paulo, Brazil Univ Estadual Campinas, Inst Chem, BR-13084862 Sao Paulo, Brazil State Univ Sao Paulo, Inst Biosci, BR-18618970 Sao Paulo, Brazil European Synchrotron Radiat Facil, CS40220, Grenoble, France State Univ Sao Paulo, Inst Biosci, BR-18618970 Sao Paulo, Brazil FAPESP: 2008/56255-9 FAPESP: 2009/52840-7 FAPESP: 2010/18773-8 FAPESP: 2013/08293-7 FAPESP: 2013/15582-5 FAPESP: 2014/10448-1 CNPq: 490022/2009-0 CNPq: 301981/2011-6 CNPq: 500091/2014-5 CNPq: 310177/2011-1

Given the current interest in second-generation biofuels, carbohydrate-active enzymes have become the most important tool to overcome the structural recalcitrance of the plant cell wall. While some glycoside hydrolase families have been exhaustively described, others remain poorly characterized, especially with regard to structural information. The family 43 glycoside hydrolases are a diverse group of inverting enzymes; the available structure information on these enzymes is mainly from xylosidases and arabinofuranosidase. Currently, only one structure of an exo-β-1,3-galactanase is available. Here, the production, crystallization and structure determination of a putative exo-β-1,3-galactanase fromBifidobacterium bifidumS17 (BbGal43A) are described.BbGal43A was successfully produced and showed activity towards synthetic galactosides.BbGal43A was subsequently crystallized and data were collected to 1.4 Å resolution. The structure shows a single-domain molecule, differing from known homologues, and crystal contact analysis predicts the formation of a dimer in solution. Further biochemical studies are necessary to elucidate the differences betweenBbGal43A and its characterized homologues.

In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with D-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with D-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two-orders of magnitude decrease of the enzyme kcat without significant modification in its Km, whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable. This article is protected by copyright. All rights reserved.

Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential.