Substrate specificity in glycoside hydrolase family 10 - Structural and kinetic analysis of the streptomyces lividans xylanase 10A

TY - JOUR

T1 - Substrate specificity in glycoside hydrolase family 10 - Structural and kinetic analysis of the streptomyces lividans xylanase 10A

AU - Ducros, V

AU - Charnock, S J

AU - Derewenda, U

AU - Derewenda, Z S

AU - Dauter, Z

AU - Dupont, C

AU - Shareck, F

AU - Morosoli, R

AU - Kluepfel, D

AU - Davies, G J

PY - 2000/7/28

Y1 - 2000/7/28

N2 - Endoxylanases are a group of enzymes that hydrolyze the beta-1,4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 Angstrom resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 Angstrom which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The Lu-linked intermediate with the nucleophile, Glu-236, is in a C-4(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

AB - Endoxylanases are a group of enzymes that hydrolyze the beta-1,4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 Angstrom resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 Angstrom which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The Lu-linked intermediate with the nucleophile, Glu-236, is in a C-4(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

KW - AMINO-ACID-SEQUENCE

KW - CRYSTAL-STRUCTURE

KW - ACTIVE-SITE

KW - ENZYME INTERMEDIATE

KW - ANGSTROM RESOLUTION

KW - MACROMOLECULAR STRUCTURES

KW - THERMOASCUS-AURANTIACUS

KW - BETA-1,4-GLYCANASE CEX

KW - CATALYTIC INTERMEDIATE

KW - MOLECULAR REPLACEMENT

M3 - Article

SN - 0021-9258

VL - 275

SP - 23020

EP - 23026

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 30

ER -