Mutant Isolation of the Escherichia coli Quinoprotein Glucose Dehydrogenase and Analysis of Crucial Residues Asp-730 and His-775 for Its Function
Copyright policy )Mutant Isolation of the Escherichia coli Quinoprotein Glucose Dehydrogenase and Analysis of Crucial Residues Asp-730 and His-775 for Its Function
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.
- Yamaguchi University Japan
Aspartic Acid, Glucose Dehydrogenases, Molecular Sequence Data, Kinetics, Amino Acid Substitution, Escherichia coli, Mutagenesis, Site-Directed, Histidine, Magnesium, Amino Acid Sequence, Sequence Alignment
Aspartic Acid, Glucose Dehydrogenases, Molecular Sequence Data, Kinetics, Amino Acid Substitution, Escherichia coli, Mutagenesis, Site-Directed, Histidine, Magnesium, Amino Acid Sequence, Sequence Alignment
citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).24 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Average influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Top 10% impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10%
Citations provided by BIP!Popularity provided by BIP!