Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered K(m) values--site-directed mutagenesis studies on the putative active site.
Based on a PCR mutant enzyme of water-soluble glucose dehydrogenase-harboring pyrroloquinoline quinone as the prosthetic group, PQQGDH-B, a site-directed mutagenesis study was carried out. The substitution of Glu277 residue with Gly resulted in a decrease in the K(m) value for glucose and altered the substrate specificity profile, compared with the wild-type enzyme. Mutational analyses on the neighboring amino acid residues of Glu277 were also carried out and constructed Asp275Glu, Asp276Glu, Ile278Phe, and Asn279His. Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca(2+), which is responsible for the binding PQQ. A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gln, or to Lys, was constructed and they all showed decreased K(m) values and altered substrate specificity profiles. Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. The potential applications of Glu277Lys in analytical use are also discussed.
Igarashi
S
S
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo, 184-8588, Japan.
Ohtera
T
T
Yoshida
H
H
Witarto
A B
AB
Sode
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004926:Escherichia coli; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone)