Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis

Abstract

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 α → α connecting loops of the catalytic (α/α)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 an ~ 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171 → Ser, Thr173 → Gly and A.niger wild-type GA had very similar k(cat) 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, T(m) was thus unchanged for Tyr175 → Phe GA and reduced by 0.2-2.9°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.