Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: Substrate distortion gives rise to the preferred axial orientation for the leaving group

TY - JOUR

T1 - Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: Substrate distortion gives rise to the preferred axial orientation for the leaving group

AU - Sulzenbacher, G

AU - Driguez, H

AU - Henrissat, B

AU - Schulein, M

AU - Davies, G J

PY - 1996/12/3

Y1 - 1996/12/3

N2 - Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysporum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, 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 presence 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 potential enzymatic cleavage is clearly distorted from the standard C-4(1) chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L. O., Johnson, L. N., Machin, P. A., Phillips, D. C., & Tijan, T. (1974) J. Mol. Biol. 88, 349-371]. The distortion observed goes beyond the ''sofa'' conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

AB - Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysporum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, 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 presence 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 potential enzymatic cleavage is clearly distorted from the standard C-4(1) chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L. O., Johnson, L. N., Machin, P. A., Phillips, D. C., & Tijan, T. (1974) J. Mol. Biol. 88, 349-371]. The distortion observed goes beyond the ''sofa'' conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

KW - ACID-SEQUENCE SIMILARITIES

KW - GLYCOSYL HYDROLASES

KW - TRICHODERMA-REESEI

KW - CRYSTAL-STRUCTURE

KW - HYDROLYSIS

KW - LYSOZYME

KW - MECHANISMS

KW - STEREOCHEMISTRY

KW - CLASSIFICATION

KW - CELLULASES

M3 - Article

SN - 0006-2960

VL - 35

SP - 15280

EP - 15287

JO - Biochemistry

JF - Biochemistry

IS - 48

ER -