REFINED STRUCTURE FOR THE COMPLEX OF 1-DEOXYNOJIRIMYCIN WITH GLUCOAMYLASE FROM ASPERGILLUS-AWAMORI VAR X100 TO 2.4-ANGSTROM RESOLUTION

The three-dimensional structure of the complex of 1-deoxynojirimycin with glucoamylase II- (471) from Aspergillus awamori var. X100 has been determined to 2.4-angstrom resolution. The model includes residues corresponding to residues 1-471 of glucoamylase I from Aspergillus niger, two molecules of bound 1-deoxynojirimycin and 605 sites for water molecules. The crystallographic R factor from refinement is 0.119, and the root-mean-squared deviation in bond distances is 0.012 angstrom. The inhibitor complex confirms the location of the active site in the packing void of the alpha/alpha-barrel as proposed by Aleshin et al. [Aleshin, A., Golubev, A., Firsov, L., & Honzatko, R. B. (1992) J. Biol. Chem. 267,19291-19298]. One inhibitor molecule is associated with strong electron density and represents the principal site of interaction of 1-deoxynojirimycin with the enyzme. The other 1-deoxynojirimycin molecule is associated with weak electron density and, therefore, probably represents a binding site of low affinity. Interactions of 1-deoxynojirimycin with the enzyme at its principal site involve Arg 45, Asp 55, Arg 305, and carbonyl 177. In addition, a water molecule (water 500) hydrogen bonds to Glu 400 and the 6-hydroxyl of 1-deoxynojirimycin and is at an approximate distance of 3.3 angstrom from the ''anomeric'' carbon of the inhibitor. The structural arrangement of functional groups near the inhibitor molecule suggests that Glu 179 is a catalytic acid, Glu 400 a catalytic base, and water 500 the attacking nucleophile in the hydrolysis of maltooligosaccharides. The relevance of the X-ray work to proposed mechanisms of enzymatic hydrolysis of oligosacchrides is discussed.