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

Endoxylanases are a group of enzymes that hydrolyze the beta-1,4-linked xyl ose backbone of xylans. They are predominantly found in two discrete sequen ce families known as glycoside hydrolase families 10 and 11. The Streptomyc es 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 Angs trom resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 2 69, 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-1 68 and Cys-201. In order to investigate substrate binding and specificity i n glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the cov alent cellobiosyl enzyme intermediates of Xyl10A were trapped through the u se of appropriate 2-fluoroglycosides. The Lu-linked intermediate with the n ucleophile, Glu-236, is in a C-4(1) chair conformation as previously observ ed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsa n, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistr y 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 s ubsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.