The K K-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid^base catalytic residue. AbfA T-6 exhibits residual activity towards aryl L L-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid^base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid^base catalyst of AbfA T-6. ß
␣-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 ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides 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 ␣-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 ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as -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 ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and -xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...
-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 -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 ϳ10 6 -fold, and this activity was enhanced 10 3 -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 Glu 335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a ). The mutant exhibited 10 3 -fold reduction in activity, and the Brønsted plot of log(k cat ) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 10 3 -fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat /K m ), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a ) but decreased 10 2 -fold for those that require strong acid catalysis (high pK a ). 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 Asp 495 acting as the acid-base in XynB2.
A L L-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65³C and pH 5.6^6.3. The stereochemistry of the hydrolysis of p-nitrophenyl L L-D-xylopyranoside was followed by 1 H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
[reaction: see text] Tuning the reactivity of glycosyl donors derived from 2-amino-2-deoxy glucose by selective introduction of different N-protecting (NPhth and NHTroc) and anomeric leaving groups (ethylthio and phenylthio) enabled highly efficient oligosaccharide synthesis in a one-pot manner. One-pot sequential glycosylation of three and four units of 2-amino-2-deoxy glucose gave trisaccharides and tetrasaccharide in 50-81% yields.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.