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 h eavily O-glycosylated sequence comprises two parts. The first (residue s 441-471) in the crystal structure wraps around an alpha/alpha-barrel formed by residues 1-440. The second (residues 472-508) is an extende d, semi-rigid linker between the two domains. To investigate the funct ional role of this linker, we made internal deletions to remove residu es 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 similar to 60 and 20% the wild-type level, re spectively, while GA Delta 1 was almost undetectable. Western blots co mparing extracellular and intracellular fractions indicated that the r egion deleted in GA Delta 3 was critical for secretion, while the regi on deleted in GA Delta 2 contributed to the production of a stable enz yme 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, i ndicating that for soluble starch their deletions did not affect the c atalytic domain and for insoluble starch the linker does not coordinat e the activities of the catalytic and starch-binding domains. The dele tions had a significant negative effect on GA Delta 2 and GA Delta 3 t hermostabilities.