STRUCTURE OF GLUCOAMYLASE FROM SACCHAROMYCOPSIS-FIBULIGERA AT 1.7 ANGSTROM RESOLUTION

The yeast Saccharomycopsis fibuligera produces a glucoamylase which be longs to sequence family 15 of glycosyl hydrolases. The structure of t he non-glycosylated recombinant enzyme has been determined by molecula r replacement and refined against 1.7 Angstrom resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-di mensional structure of a yeast family 15 glucoamylase. There refinemen t from the initial molecular-replacement model was not straightforward . It involved the use of an unrestrained automated refinement procedur e (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alp ha-helices, 12 of which form an (alpha/alpha)(6) barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of t he catalytic domain of a fungal family 15 glucoamylase and are shown t o be closely similar. The active- and specificity-site residues are es pecially highly conserved. The model of the acarbose inhibitor from th e analysis of the fungal enzyme fits tightly into the present structur e, The active-site topology is a pocket and hydrolysis proceeds with i nversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethy l)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.