Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution.
The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-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 the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
J
J
Sevcík
A
A
Solovicová
E
E
Hostinová
J
J
Gasperík
K S
KS
Wilson
Z
Z
Dauter
D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015003:Yeasts / Q000201:enzymology*
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; D005087:Glucan 1,4-alpha-Glucosidase