Characterization of BrGH3A, a bovine rumen-derived glycoside hydrolase family 3 ß-glucosidase with a permuted domain arrangement.

The bovine rumen contains a large consortium of residential microbes that release a variety of digestive enzymes for feed degradation. However, the utilization of these microbial enzymes is still limited because these rumen microorganisms are mostly anaerobes and are thus unculturable. Therefore, we applied a sequence-based metagenomic approach to identify a novel 2,445-bp glycoside hydrolase family 3 ß-glucosidase gene known as BrGH3A from the metagenome of bovine ruminal fluid. BrGH3A ß-glucosidase is a 92-kDa polypeptide composed of 814 amino acid residues. Unlike most glycoside hydrolases in the same family, BrGH3A exhibited a permuted domain arrangement consisting of an (α/ß)6 sandwich domain, a fibronectin type III domain and a (ß/α)8 barrel domain. BrGH3A exhibited greater catalytic efficiency toward laminaribiose than cellobiose. We proposed that BrGH3A is an exo-acting ß-glucosidase from Spirochaetales bacteria that is possibly involved in the intracellular degradation of ß-1,3-/1,4-mixed linkage glucans that are present in grass cell walls. BrGH3A exhibits rich diversity in rumen hydrolytic enzymes and may represent a member of a new clan with a permuted domain topology within the large family.

Structural and mutational analysis of glycoside hydrolase family 1 Br2 ß-glucosidase derived from bovine rumen metagenome.

Ruminant animals rely on the activities of ß-glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 ß-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (ß/α)8-TIM barrel domain but displays unique structural features at loop ß5→α5 and α-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in ß-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii ß-glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 ß-glucosidases and may provide a basis for future enzyme engineering applications.

Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX ß-xylosidase from Aspergillus niger.

ß-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 ß-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (ß/α)8 TIM-barrel-like domain, an (α/ß)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.

Synthesis of long-chain alkyl glucosides via reverse hydrolysis reactions catalyzed by an engineered ß-glucosidase.

Long-chain alkyl glucosides, such as octyl and decyl ß-d-glucopyranosides (OG and DG, respectively), are regarded as a new generation of biodegradable, non-ionic surfactants. Previously, the mutants of Dalbergia cochinchinensis Pierre dalcochinase showed potential in the synthesis of oligosaccharides and alkyl glucosides. In this study, the N189F dalcochinase mutant gave the highest yields of OG and DG synthesis under reverse hydrolysis conditions. The optimized yield of OG (57.5 mol%) was obtained in the reactions containing 0.25 M glucose and 0.3 units of the N189 F mutant in buffer-saturated octanol at 30 °C. The identity of OG and DG products was confirmed by high resolution mass spectrometry (HRMS) and NMR. Consistent with its capability for synthesis, the reactivation kinetics and ITC analysis revealed that the aglycone binding pocket of the N189F mutant was more favorable for long-chain alkyl alcohols than the wild-type dalcochinase, while their glycone binding pockets showed similar affinity for the glucosyl moiety. STD NMR revealed higher interactions at the aglycone sites than the glycone sites. Our results demonstrated a promising potential of the N189F dalcochinase mutant in the future commercial production of long-chain alkyl glucosides via reverse hydrolysis reactions.

Improved synthesis of long-chain alkyl glucosides catalyzed by an engineered ß-glucosidase in organic solvents and ionic liquids.

OBJECTIVE: To synthesize octyl ß-D-glucopyranoside (OG) and decyl ß-D-glucopyranoside (DG) in three non-aqueous reaction systems, namely organic solvents, ionic liquids and co-solvent mixtures, via reverse hydrolysis reactions catalyzed by the N189F dalcochinase mutant. RESULTS: The highest yield of OG (67 mol%) was obtained in the reaction containing 0.5 M glucose, 3 unit ml-1 enzyme in 20% (v/v) octanol and 70% (v/v) [BMIm][PF6] at 30 °C. On the other hand, the highest yield of DG (64 mol%) was obtained in the reaction containing 0.5 M glucose, 3 unit ml-1 enzyme in 20% (v/v) decanol, 20% (v/v) acetone and 50% (v/v) [BMIm][PF6] at 30 °C. The identities of OG and DG products were confirmed by HRMS and NMR. CONCLUSION: This is the first report of enzymatic synthesis of OG and DG via reverse hydrolysis reactions in ionic liquids and co-solvent mixtures. The N189F dalcochinase mutant and the non-aqueous reaction systems described here show great potential for future commercial production of long-chain alkyl glucosides.

Multiple mutations in the aglycone binding pocket to convert the substrate specificity of dalcochinase to linamarase.

Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are ß-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol. So, the space provided by the I185A mutation appeared to be a prerequisite for accommodation of the aglycone moiety containing three substituents at the carbinol carbon. However, quantitative analysis of the energy parameters revealed mostly antagonistic interactions between these mutations. In addition, the N189F mutant showed a potential for use in enzymatic synthesis of alkyl glucosides via transglucosylation and reverse hydrolysis reactions. Thus, substitution of only 2-3 key residues in the aglycone binding pocket of dalcochinase could convert its specificities to that of linamarase, as well as to be suitable for any chosen hydrolytic or synthetic applications.

Molecular Characterization and Potential Synthetic Applications of GH1 ß-Glucosidase from Higher Termite Microcerotermes annandalei.

A novel ß-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting ß-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) ß-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-ß-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 ß-glucosidase and its applications in bioconversion and biotechnology.

Cloning, expression and characterization of ß-xylosidase from Aspergillus niger ASKU28.

ß-Xylosidases catalyze the breakdown of ß-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 ß-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the ß-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 ß-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.

Purification and characterization of three ß-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28.

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. ß-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three ß-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely ß-xylosidase (P1.1), ß-glucosidase (P1.2), and glucan 1,3-ß-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial ß-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant ß-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.

Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis ß-glucosidase to increase catalytic efficiency toward mannosides.

Dalcochinase and Abg are glycoside hydrolase family 1 ß-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl ß-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer ß-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl ß-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.

Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-ß-glucosidase SPy1599.

The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α-1,6- and GH38 α-1,3-mannosidases (SPy1603 and SPy1604), a GH84 ß-hexosaminidase (SPy1600) and a putative GH2 ß-galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative ß-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (ß/α)(8)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a ß-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-ß-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-ß-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) beta-glucosidase from Aspergillus niger ASKU28.

BACKGROUND: The commercially important glycoside hydrolase family 3 (GH3) beta-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined. METHODS: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 beta-glucosidase from A. niger ASKU28. RESULTS: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K(m) values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl beta-glucosides and yielded beta-glucosyl azide as a product. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results strongly support the assignment of E490 as the acid/base catalyst in a beta-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.

Functional and structural differences between isoflavonoid beta-glycosidases from Dalbergia sp.

Among isoflavonoid beta-glucosidases from Dalbergia species, that from Dalbergia nigrescens hydrolyzes isoflavonoid-7-O-beta-D-apiosyl-1,6-beta-D-glucosides more efficiently, while Dalbergia cochinchinensis beta-glucosidase (dalcochinase) hydrolyzes its rotenoid glycoside substrate, dalcochinin beta-d-glucoside (I), more efficiently. A cDNA encoding a glycosylated beta-glucosidase with 81% identity with dalcochinase was cloned from D. nigrescens seeds, and its protein (Dnbglu2) expressed in Pichia pastoris. Purified Dnbglu2 hydrolyzed the D. nigrescens natural substrates dalpatein 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (II) and dalnigrein 7-O-beta-d-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (III) at 400- and 5000-fold higher catalytic efficiency (k(cat)/K(m)) than I. Dalcochinase was mutated at two amino acid residues, A454S and E455G, that are homologous to previously described substrate binding residues and differ from the corresponding residues in Dnbglu2. The double mutant showed 4- and 6.8-fold increases in relative activity toward II and III, respectively. However, this activity was only 3% that of Dnbglu2 beta-glucosidase, indicating other determinants are important for isoflavonoid diglycoside hydrolysis.