Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase.
Aspergillus awamori glucoamylase (GA) contains globular catalytic and starch-binding domains (residues 1-471 and 509-616, respectively). A heavily O-glycosylated sequence comprises two parts. The first (residues 441-471) in the crystal structure wraps around an alpha/alpha-barrel formed by residues 1-440. The second (residues 472-508) is an extended, semi-rigid linker between the two domains. To investigate the functional role of this linker, we made internal deletions to remove residues 466-512 (GA delta 1), 485-512 (GA delta 2) and 466-483 (GA delta 3). GA delta 2 and GA delta 3 were expressed in Saccharomyces cerevisiae culture supernatants at approximately 60 and 20% the wild-type level, respectively, while GA delta 1 was almost undetectable. Western blots comparing extracellular and intracellular fractions indicated that the region deleted in GA delta 3 was critical for secretion, while the region deleted in GA delta 2 contributed to the production of a stable enzyme structure. The activities of purified GA delta 2 and GA delta 3 on soluble and insoluble starch were similar to those of wild-type GA, indicating that for soluble starch their deletions did not affect the catalytic domain and for insoluble starch the linker does not coordinate the activities of the catalytic and starch-binding domains. The deletions had a significant negative effect on GA delta 2 and GA delta 3 thermostabilities.
Department of Chemistry, Colby College, Waterville, ME 04901.
C B
CB
Libby
C A
CA
Cornett
P J
PJ
Reilly
C
C
Ford
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015202:Protein Engineering; D017384:Sequence Deletion; D013213:Starch; D013379:Substrate Specificity; D013816:Thermodynamics
D004271:DNA, Fungal; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase