Deletion analysis of the starch-binding domain of Aspergillus glucoamylase.
The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.
Chen
L
L
Department of Zoology, Iowa State University, Ames 50011, USA.
Coutinho
P M
PM
Nikolov
Z
Z
Ford
C
C
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D013213:Starch; D013696:Temperature
D004271:DNA, Fungal; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase