Cb. Libby et al., EFFECT OF AMINO-ACID DELETIONS IN THE O-GLYCOSYLATED REGION OF ASPERGILLUS-AWAMORI GLUCOAMYLASE, Protein engineering, 7(9), 1994, pp. 1109-1114
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.