THE TOPOLOGY OF THE SUBSTRATE-BINDING CLEFTS OF GLYCOSYL HYDROLASE FAMILY 10 XYLANASES ARE NOT CONSERVED

The crystal structures of family 10 xylanases indicate that the distal regions of their active sites are quite different, suggesting that th e topology of the substrate binding clefts of these enzymes may vary. To test this hypothesis, we have investigated the rate and pattern of xylooligosaccharide cleavage by the family 10 enzymes, Pseudomonas flu orescens subsp. cellulosa xylanase A (XYLA) and Cellulomonas fimi exog lucanase, Cex. The data showed that Cex contained three glycone and tw o aglycone binding sites, while XYLA had three glycone and four aglyco ne binding sites, supporting the view that the topologies of substrate binding clefts in family 10 glycanases are not highly conserved. The importance of residues in the substrate binding cleft of XYLA in catal ysis and ligand binding were evaluated using site-directed mutagenesis . In addition to providing insight into the function of residues in th e glycone region of the active site, the data showed that the aromatic residues Phe-181, Tyr-255, and Tyr-220 play important roles in bindin g xylose moieties, via hydrophobic interactions, at subsites +1, +3, a nd +4, respectively. Interestingly, the F181A mutation caused a much l arger reduction in the activity of the enzyme against xylooligosacchar ides compared with xylan. These data, in conjunction with a previous s tudy (Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G;. P., Pickersgill, R., and Gilbert, H. J. (1997) J . Biol. Chem. 272, 2942-2951), suggest that the binding of xylooligosa ccharides at the -2 and +1 subsites ensures that the substrates occupy the -1 and +1 subsites and thus preferentially form productive comple xes with the enzyme. Loss of ligand binding at either subsite results in small substrates forming nonproductive complexes with XYLA by bindi ng to distal regions of the substrate binding cleft.