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 Escheri chia coli were obtained and characterized. Of these, significant mutan ts were further characterized by kinetic analysis after purification o r by site-directed mutagenesis to introduce different amino acid subst itutions. H775R and H775A showed a pronounced reduction of affinity fo r a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His775 may directly interact with PQQ, D730N and D730A showed low gluc ose 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 surroundi ng PQQ was not changed by D730N mutation. Based on these data, we assu me that Asp-730 may occur close to PQQ and function as a proton (and a lso electron) donor to PQQ or acceptor from PQQH(2). Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affi nity for PQQ or GDH activity. Therefore, the unique segment and Asp-73 0 may play a specific role for GDH, which might be related to the intr amolecular electron transfer from PQQ to ubiquinone.