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PubMed1998

Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase.

The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism. A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class. Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion. Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation. Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.

Bacillus↗
PubMed2003

The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.

beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

Amino Acid Sequence↗
PubMed2002

Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.

alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.

Alanine↗
PubMed2001

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase.

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Deltaabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Deltaabf51A against aryl-alpha-arabinofuranosides, arabinan and alpha1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

Blotting, Southern↗
PubMed2002

Identification of residues important for NAD+ binding by the Thermotoga maritima alpha-glucosidase AglA, a member of glycoside hydrolase family 4.

The NAD+-requiring enzymes of glycoside hydrolase family 4 (GHF4) contain a region with a conserved Gly-XXX-Gly-Ser (GXGS) motif near their N-termini that is reminiscent of the fingerprint region of the Rossmann fold, a conserved structural motif of classical nicotinamide nucleotide-binding proteins. The function of this putative NAD+-binding motif in the alpha-glucosidase AglA of Thermotoga maritima was probed by directed mutagenesis. The K(d) for NAD+ of the AglA mutants G10A, G12A and S13A was increased by about 300-, 5-, and 9-fold, respectively, while their K(m) for p-nitrophenyl-alpha-glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD+ binding by the glycosidases of GHF4.

Amino Acid Motifs↗
PubMed1979

A glycoside-hydrolase inhibitor in treatment of dumping syndrome.

BAY g 5421, a glycoside-hydrolase inhibitor, produced symptomatic improvement in ten patients with the dumping syndrome. 100 mg BAY g 5421, given before a 50 g sucrose meal, produced pronounced attenuation of both hyperglycaemic and hypoglycaemic phases of plasma glucose levels; and it greatly reduced the rise in plasma levels of gastric inhibitory polypeptide and insulin. Gastric emptying, studied simultaneously by an isotopic method, showed little difference between tests, suggesting that the improvement achieved was not mediated by slowing gastric emptying.

Adult↗
PubMed2004

Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68.

Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665-668] and Bacillus subtilis endo-1,5-alpha-L-arabinanase. A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction.

Amino Acid Sequence↗
PubMed2003

Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Brønsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.

Bacillaceae↗
PubMed2025

Mining metagenomes from extremophiles as a resource for novel glycoside hydrolases for industrial applications.

The exploration of metagenomes from extremophiles has emerged as a promising approach for discovering novel glycoside hydrolases (GHs) with potential industrial applications. Extremophiles, which thrive in harsh conditions such as high salinity, extreme temperatures, and acidic or alkaline environments, produce enzymes naturally adapted to function under these conditions. This unique adaptability makes them highly desirable for industrial processes requiring robust and efficient biocatalysts. These biocatalysts reduce reliance on harsh chemicals and energy-intensive processes, contributing to greener industrial operations. This review underscores the power of metagenomics in bypassing the need to culture large libraries of extremophiles in the lab. High-throughput sequencing and bioinformatics enable the identification of novel GH-encoding genes directly from environmental DNA. While metagenomic mining has yielded promising results, challenges such as the expression of extremophile-derived genes in mesophilic hosts, low activity yields, and scalability remain. Advances in synthetic biology and protein engineering could address these bottlenecks, enabling more efficient utilization of GHs. Additionally, integrating machine learning for predictive functional annotation may accelerate the identification of high-value candidates.

Glycoside Hydrolases↗
PubMed2000

Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis.

Lysosomal storage diseases are due to inherited deficiencies in various enzymes involved in basic metabolic processes. As with other genetic diseases, accurate structure data for these enzymatic proteins should help in better understanding the molecular effects of mutations identified in patients with the corresponding lysosomal diseases; however, no such three-dimensional (3D) structure data are available for many lysosomal enzymes. Thus, we herein intend to illustrate for an audience of molecular geneticists how structure information can nonetheless be obtained via a bioinformatics approach in the case of five human lysosomal glycoside hydrolases. Indeed, using the two-dimensional hydrophobic cluster analysis method to decipher the sequence information available in data banks for the large group of glycoside hydrolases (clan GH-A) to which these human lysosomal enzymes belong, we could deduce structure predictions for their catalytic domains and propose explanations for the molecular effects of mutations described in patients. In addition, in the case of human beta-glucuronidase for which experimental 3D data have been reported, we also show here that bioinformatics methods relying on the available 3D structure information can be used to obtain further insights into the effects of various mutations described in patients with Sly disease. In a broader perspective, our work stresses that, in the context of a rapid increase in protein sequence information through genome sequencing, bioinformatics approaches might be highly useful for generating structure-function predictions based on sequence-structure interrelationships.

Crystallography, X-Ray↗
PubMed2001

The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases.

BACKGROUND: kappa-carrageenans are gel-forming, sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of marine red algae. The kappa-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades kappa-carrageenan both in solution and in solid state by an endoprocessive mechanism. This beta-galactanase belongs to the clan-B of glycoside hydrolases. RESULTS: The structure of P. carrageenovora kappa-carrageenase has been solved to 1.54 A resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme. The enzyme folds into a curved beta sandwich, with a tunnel-like active site cavity. Another remarkable characteristic is the presence of an arginine residue at subsite -1. CONCLUSIONS: The crystal structure of P. carrageenovora kappa-carrageenase is the first three-dimensional structure of a carrageenase. Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides. Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral beta bulge, and the other in which it is held by a regular beta strand. At subsite -1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis. In addition, in kappa-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the beta-linked kappa-carrageenan monomers. It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.

Alteromonas↗
PubMed2002

The 1.62 A structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5.

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 A resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold beta/alpha barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites -4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite -1.

Ascomycota↗
PubMed2002

An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2.

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

Amino Acid Sequence↗
PubMed2002

Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose.

The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively. A4-beta-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.

Amino Acid Sequence↗
PubMed2003

Identification and analysis of catalytic TIM barrel domains in seven further glycoside hydrolase families.

Fold recognition results allocate catalytic triose phosphate isomerase (TIM) barrels to seven previously unassigned glycoside hydrolase (GH) families, numbers 29, 44, 50, 71, 84, 85 and 89, enabling prediction of catalytic residues. Modelling of GH family 50 suggests that it may be the common evolutionary ancestor of families 42 and 14. TIM barrels now comprise the catalytic domains of more than half of the assigned GH families, and catalyse a much larger variety of GH reactions than any other catalytic domain architecture. Only 327 GH sequences still have no structurally identified catalytic domain.

Amino Acid Sequence↗
PubMed2003

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.

Amino Acid Sequence↗
PubMed2002

Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13.

Classification of glycoside hydrolases (GHs) into families, along with the structure-based grouping together of families into clans, improve our understanding of the evolution of the large natural variety of these enzymes, help rationalise experimental data and guide further studies. Here we identify triose phosphate isomerase (TIM) barrels in GH families 27, 31, 36 and 66. We further show that iterated sequence database searches provide evidence for their sharing a common evolutionary origin with GH family 13. The catalytic, nucleophilic residue common to all these families is thereby determined and candidate catalytic proton donors identified within each family.

Aspartic Acid↗