Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis.
To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.
Stoffer
B B
BB
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Dupont
C
C
Frandsen
T P
TP
Lehmbeck
J
J
Svensson
B
B
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D004795:Enzyme Stability / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000502:physiology*; D015202:Protein Engineering; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship
D005087:Glucan 1,4-alpha-Glucosidase