STRUCTURE OF THE FUSARIUM-OXYSPORUM ENDOGLUCANASE-I WITH A NONHYDROLYZABLE SUBSTRATE-ANALOG - SUBSTRATE DISTORTION GIVES RISE TO THE PREFERRED AXIAL ORIENTATION FOR THE LEAVING GROUP

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall reten tion of configuration. The structure of the EG I from Fusarium oxyspor um, complexed to a nonhydrolyzable thiooligosaccharide substrate analo gue, has been determined by X-ray crystallography at a resolution of 2 .7 Angstrom utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the p resence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potent ial enzymatic cleavage is clearly distorted from the standard C-4(1) c hair as was originally suggested for beta-retaining enzymes by Phillip s [Ford, L. O., Johnson, L. N., Machin, P. A., Phillips, D. C., & Tija n, T. (1974) J. Mol. Biol. 88, 349-371]. The distortion observed goes beyond the ''sofa'' conformation observed in previous studies and resu lts in a conformation whose salient feature is the resulting quasi-axi al orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has rece ntly been observed in a complex of chitobiase with its unhydrolyzed su bstrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The strik ing similarity between these two complexes extends beyond the almost i dentical pyranose ring distortion. The overlap of the two respective s ugars places the enzymatic nucleophile of endoglucanase I in coinciden ce with the C2 acetamido oxygen of N-acetylglucosamine in the catalyti c site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enz ymes. The endoglucanase I complex with the thiosaccharide substrate an alogue clearly illustrates the potential of nonhydrolyzable sulfur-lin ked oligosaccharides in the elucidation of substrate binding and catal ysis by glycosyl hydrolases.