Abstract
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.
Highlights
Quinoprotein GDH1 bound to the inner membrane in Escherichia coli functions in a direct oxidation of D-glucose to Dgluconate and concomitantly transferring the electrons to cytochrome oxidase through ubiquinone in the respiratory chain [1, 2]
The ubiquinone-binding site has been indicated to be at the region close to the periplasmic side by using reconstituted proteoliposomes [10], where no membrane potential is generated by the electron transfer from glucose to ubiquinone in the dehydrogenase
Isolation of the gcd Mutants—Out of 30 GcdϪ mutants isolated from region-specific mutagenesis, targeting the conserved C terminus of glucose dehydrogenase (GDH), four were found to exhibit less than 10% PMS reductase activity of the wild type
Summary
Materials—Restriction enzymes, T4 DNA ligase, Taq DNA polymerase, and the DNA sequencing kit were purchased from Takara Shuzo (Kyoto, Japan). The EcoRI-PstI fragment bearing the gcd gene from pUCGCD1 was inserted into the EcoRI-PstI site of pACYC177–322, generating pACGCD1. From the resulting pUCGCD2, the EcoRI-PstI fragment bearing the gcd gene was inserted into the EcoRI-PstI site of pACYC177-322, generating pACGCD2. Each mutated region of the gcd gene was replaced into the wild type gcd gene of pUCGCD1 to facilitate enzyme assay. Screening of the gcd Mutants—Mutagenized plasmid DNA was introduced into YU312, and weak red color colonies on tetrazolium plates [32] containing 0.5% glucose, 0.1 M PQQ, and 50 g/ml kanamycin were isolated as the gcd mutant. To determine the region including the mutation site of the mutants from hydroxylamine mutagenesis, recombination between the mutant gcd genes and the wild type gcd gene was performed. The purified protein was readsorbed to DEAE column equilibrated with 10 mM potassium phosphate, pH 7.0, containing 0.1% Triton X-100, and after washing with the same buffer in the absence of Triton, the enzyme was eluted with the same buffer containing 0.2% -octylgluoside
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