Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

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 . 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).Membrane-bound quinoprotein GDH of Escherichia coli catalyzes oxidation of the C-1 hydroxyl group of the pyranose form of D-glucose to D-glucono-␦-lactone, which is spontaneously converted to D-gluconate, and concomitantly transfers electrons to ubiquinol oxidase through ubiquinone in the respiratory chain (2, 3). Topological analysis revealed that the monomeric GDH possesses five trans-membrane segments at the N-terminal portion (residues 1-154), which ensure strong anchorage of the protein in the inner membrane (4). The remaining C-terminal portion (residues 155-796) occurs at the periplasmic side of the membrane. This portion is assumed to have a catalytic domain including PQQ (5, 6) and Ca 2ϩ or Mg 2ϩ binding sites (7,8). Moreover, GDH of E. coli occurs as an apoenzyme (7, 8), and the exogenous addition of PQQ with the divalent cation leads to formation of the active enzyme (9).Three-dimensional structures of MDHs from three different bacteria have been determined by x-ray crystallography (10 -12), which reveals that the ␣ subunit is a superbarrel made up of eight topologically identical four stranded anti-parallel ␤ sheets, being arranged with radial symmetry like the blades of a propeller. PQQ is tightly stacked within a chamber of the active site in the ␣ subunit, and Ca 2ϩ helps PQQ to be maintained in the correct configuration. Amino acid residues interacting with PQQ and Ca 2ϩ are dispersed in the whole ␣ subunit.Alignment of the PQQ-binding proteins or subunits among quinoprotein dehydrogenases reveals that the periplasmic domain of GDH in E. coli has 26% sequence similarity to the ␣ subunit of MDH...

Amino Acid Residues Interacting with Both the Bound Quinone and Coenzyme, Pyrroloquinoline Quinone, in Escherichia coli Membrane-bound Glucose Dehydrogenase

“…In this study, we identified amino acid residues interacting with bound UQ and proposed their functions in intramolecular events of the enzyme. Clues for this issue have been obtained by previous studies that revealed very close location of the bound UQ to PQQ (31) and the position and functions of Asp-466 and Lys-493 flanking PQQ (11). Site-directed mutants of Asp-354, Asp-466, and Lys-493 had significant effects on the characteristics of UQ semiquinone radicals or UQ EPR signals and/or the affinity for UQ.…”

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…6). Pulse radiolysis analysis revealed that the two redox centers are closely located at a distance of 11-13 Å and that Asp466 and Lys493 are involved in proton donation to the semiquinone anion radical of bound Q and in electron transfer from bound UQ to PQQ, respectively (Elias et al, 2000;Mustafa et al, 2008a). Recent mGDH analysis provided the first evidence that the primary dehydrogenase in respiratory chains utilizes both MK and UQ as a bound Q and suggest that bound MK occurs in a fashion similar to that of bound UQ in the mGDH molecule and functions as an electron acceptor from PQQ (Mustafa et al, 2008b).…”

C-terminal Periplasmic Domain of Escherichia coli Quinoprotein Glucose Dehydrogenase Transfers Electrons to Ubiquinone

“…Glucose oxidase activity of membrane fractions (coupling ability of GDH to an electron transport chain in the membrane) was determined using an oxygen electrode as described previously (13,32). Q-2 reductase activity was measured spectrophotometrically as described previously (13). One unit of PMS reductase or Q-2 reductase activity is defined as 1 mol of dichloroindophenol or Q-2, respectively, reduced/ min, both of which correspond to 1 mol of glucose oxidized/min.…”

“…Using the holo-enzyme thus prepared, the following enzyme activities were measured. PMS reductase activity was measured spectrophotometrically (U-2000A, Hitachi) with PMS and dichloroindophenol as an electron mediator and acceptor, respectively, as described previously (13,32). Glucose oxidase activity of membrane fractions (coupling ability of GDH to an electron transport chain in the membrane) was determined using an oxygen electrode as described previously (13,32).…”

“…The remaining large C-terminal portion (residues 141-796) has a catalytic domain including PQQ-(6, 7) and Ca 2ϩ or Mg 2ϩ -binding sites (8,9) that is located in the periplasmic side. A model structure of GDH based on the x-ray crystallographic structure of the ␣-subunit of MDH in Methylobacterium extorquens has been proposed (10), and the putative structure of the PQQ-binding catalytic site has been further confirmed and characterized by mutagenic analysis of several amino acid residues around PQQ (11)(12)(13)(14)(15).…”