Abstract
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).
Highlights
PQQ1 is a non-covalently bound prosthetic group of most quinoprotein dehydrogenases in Gram-negative bacteria, which are involved in the oxidation of alcohols or aldose sugars in their periplasm (1)
glucose dehydrogenase (GDH) of E. coli occurs as an apoenzyme (7, 8), and the exogenous addition of pyrroloquinoline quinone (PQQ) with the divalent cation leads to formation of the active enzyme (9)
The PQQ in GDH is proposed to be sandwiched between Trp-404 and His-262 by analogy with the MDH structure in M. extorquens (13), where PQQ is tightly stacked between Trp-243 and a novel disulfide ring composed of Cys-103 and Cys-104 (17)
Summary
Materials—All restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from Takara Shuzo (Kyoto, Japan). Oligonucleotide primers for site-specific mutagenesis were purchased from Sawady Technology (Tokyo, Japan). Mutagenesis—To construct all mutants of GDH, site-specific mutagenesis was carried out using the Mutan-Super Express Km kit (Takara Shuzo, Japan). Expression of GDH mutants was examined by SDS-polyacrylamide gel electrophoresis, followed by Western blotting using a polyclonal antibody raised against E. coli GDH as described previously (30). Critical mutant GDHs as well as the wild-type GDH were purified according to the procedure described (4, 19).
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