Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Abstract

␤-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.

Multiple Regulatory Mechanisms Control the Expression of the Geobacillus stearothermophilus Gene for Extracellular Xylanase

“…The expression of the extracellular xylanase provides the cell with ample amounts of decorated xylo-oligosaccharides that enter the cell via two dedicated ABC sugar transporters, XynEFG for xylooligosaccharides and AguEFG for aldotetrauronic acid. The decorated xylooligomers are hydrolyzed to their corresponding monomers by intracellular side chain-cleaving enzymes, including ␣-glucuronidase (Agu67A) (35,39), two ␣-L-arabinofuranosidases (Abf51A and Abf51B) (36), two xylan acetylesterases (CE4) (31,42), an intracellular xylanase (Xyn10A) (43), and three ␤-xylosidases (Xyn39B, Xyn52B2, and Xyn43B3) (33,34,37,38,41). The extracellular xylanase gene (xynA) is subjected to carbon catabolite repression mediated by the global negative repressor CcpA and most likely by CodY.…”

“…The structure-function relationships of many of its hemicellulolytic enzymes were investigated intensively (31)(32)(33)(34)(35)(36)(37)(38)(39) together with its arabinan-and galactandegrading systems (19,20). The bacterium possesses two-and three-component oligosaccharide-sensing systems that activate the expression of dedicated ABC sugar transporters (19 -22, 40), allowing the cell to rapidly respond to very low carbohydrates concentrations in the soil and to efficiently take up the oligosaccharides (20,21).…”

“…When grown in the presence of xylan, strain T-6 secretes a single endo-1,4-␤-extracellular xylanase (Xyn10A) that hydrolyzes the natural polymer's main backbone, producing short xylo-oligosaccharides decorated with various side chains, such as L-arabinose, 4-O-methylglucuronic acid, or acetate. These xylosaccharides enter the cell via specialized ABC sugar transporters and are hydrolyzed to xylose monomers by intracellular enzymes, including ␣-glucuronidase (Agu67A) (35,39), two ␣-L-arabinofuranosidases (Abf51A and Abf51B) (36), three ␤-xylosidases (Xyn39B, Xyn52B2, and Xyn43B3) (33,34,37,38,41), two xylan acetylesterases (CE4) (31,42), and an intracellular xylanase (Xyn10A2) (Fig. 1) (43).…”

A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides inGeobacillus stearothermophilus

“…When grown in the presence of xylan, strain T-6 secretes a single extracellular endo 1,4-␤ xylanase (XynA) that hydrolyzes the polymer's main backbone, producing short modified oligoxylose units of two or more sugars in length. These modified xylosaccharides enter the cell by specialized ATP-binding cassette (ABC) sugar transporters (73), and they are further degraded to monomers by intracellular hydrolases, including a glycoside hydrolase family 10 (GH10) xylanase (77), two GH51 ␣-L-arabinofuranosidases (29), an ␣-glucuronidase (GH67) (23,68,89), three ␤-xylosidases (GH39, GH43, and GH52) (6,(8)(9)(10)(11), and two xylan acetyl esterases (CE4).…”

A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases

“…1, f and g) (31)(32)(33). In this approach, definite identification of the acid/base and nucleophile can be achieved by determining the azidoproduct stereochemistry via NMR.…”