Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.     

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 Br?nsted 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.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6

Beta-D-xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides into xylose units. Here, we describe the characterization and kinetic analysis of a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3). Enzymes in this family use an inverting single-displacement mechanism with two conserved carboxylic acids, a general acid, and a general base. XynB3 was most active at 65 degrees C and pH 6.5, with clear preference to xylose-based substrates. Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the nonreducing end of xylooligomers. On the basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as the general-base and general-acid catalytic residues, respectively. Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pK(a) of the leaving group. The pH-dependence activity profile of the putative general-acid mutant (E187G) revealed that the protonated catalytic residue was removed. Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant. On the basis of thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule. Together, these results confirm the suggested catalytic role of Glu187 and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact roles of the catalytic residues in an inverting GH43 glycosidase.     

Catalytic mechanism of beta-amylase from Bacillus cereus var. mycoides: chemical rescue of hydrolytic activity for a catalytic site mutant (Glu367-->Ala) by azide

The hydrolytic activity of beta-amylase from Bacillus cereus var. mycoides was lost on replacement of either of the catalytic residues (Glu172 or Glu367) with an alanyl residue. When maltopentaose and 2 M azide existed together mutant, E367A cleaved the glucosidic linkage of maltopentaose and produced maltose at pH 7.0 and 25 degrees C, but the other mutants (E172A and double mutant E172A/E367A) did not. This indicates that azide acts as a general base instead of E367 and Glu172 acting as general acids, and that the hydroxide ion generated from a water molecule activated by azide attacks a reactive pyranose nucleophilically so that beta-maltose is produced.     

Probing the catalytically essential residues of the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus

The alpha-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of alpha-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu(28), Glu(176) and Glu(298) are important for catalytic activity. Kinetic data obtained for the mutant Glu(176)-->Gln, combined with the results of chemical rescue using the mutant Glu(176)-->Ala, have shown that Glu(176) is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu(176)-->Ala, indicated that alpha-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu(298) might be the catalytic nucleophile and that Glu(28) is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that alpha-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.     

Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue.

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.     

Identification of the catalytic acid base residue of arthrobacter endo-beta-N-acetylglucosaminidase by chemical rescue of an inactive mutant

Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyses the hydrolysis and transglycosylation of asparagine-linked oligosaccharides of glycoproteins with retention of anomeric configuration. Glu-173 of Endo-A is a catalytically essential amino acid residue, and the corresponding residue is conserved in all GH family 85 members. The catalytic activity of Endo-A E173A mutant was rescued by the addition of sodium azide or sodium formate. Furthermore, the produced beta-glycosyl azide (Man(5)GlcNAc-beta-N(3)) retained the anomeric configuration, indicating that Glu-173 is the catalytic acid-base residue of Endo-A. This is the first identification of the catalytic residue for GH family 85 endo-beta-N-acetylglucosaminidases.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes

The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Br?nsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.     

Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase.

The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.     

Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases

The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.     

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0–5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Cloning and characterization of Thermotoga maritima beta-glucuronidase

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.     

X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.     

Characterization of a Hexameric Exo-Acting GH51 α-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

α-l-Arabinofuranosidases (α-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 °C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of α/β proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 °C, which is in agreement with the temperature–activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the β-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dissection of nucleophilic and acid-base catalysis in glycosidases.

A startling array of added anions have been observed to function as replacement catalytic nucleophiles in mutant glycosidases, including formate, azide, fluoride and other halides. Likewise, the mechanism of acid-base catalysis is somewhat plastic. The carboxylic acids can be substituted by a sulfenic acid or by ascorbate, and the effective acid strength enhanced by the introduction of strong hydrogen bonds. These studies provide an interesting bridge between enzymes and models thereof.