J. Sevcik et al., STRUCTURE OF GLUCOAMYLASE FROM SACCHAROMYCOPSIS-FIBULIGERA AT 1.7 ANGSTROM RESOLUTION, Acta crystallographica. Section D, Biological crystallography, 54, 1998, pp. 854-866
Citations number
53
Categorie Soggetti
Crystallography,"Biochemical Research Methods",Biophysics,Biology
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.