Spectral and kinetic studies were performed on enzyme forms of soluble glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in which the PQQ-activating Ca(2+) was absent (Holo X) or was replaced with Ba(2+) (Ba-E) or in which PQQ was replaced with an analogue or a derivative called "nitroPQQ" (E-NPQ). Although exhibiting diminished rates, just like sGDH, all enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron acceptor. On inspection of the plots for the reductive half-reaction, it appeared that the enzyme forms exhibited a negative cooperativity effect similar to that of sGDH itself under turnover conditions, supporting the view that simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E with glucose showed a fluorescing transient previously observed in the reaction of sGDH with glucose-1-d, whereas no intermediate was detected at all in the reactions of E-NPQ and Holo X. Using hydrazine as a probe, the fluorescing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Holo X, but E-NPQ did not react with hydrazine. When this is combined with other properties of E-NPQ and the behavior of enzyme forms containing a PQQ analogue, we concluded that the catalytic potential of the cofactor in the enzyme is not determined by its adduct-forming ability but by whether it is or can be activated with Ca(2+), activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the reduced cofactor in the enzyme. This conclusion, together with the observed deuterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and that already known on transient decay, indicate that the sequential steps in the mechanism of sGDH must be (1) reversible substrate binding, (2) direct transfer of a hydride ion (reversible or irreversible) from the C1 position of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-determining tautomerization of the fluorescing, C5-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxidation of PQQH(2) by an electron acceptor. The PQQ-activating Ca(2+) greatly facilitates the reactions occurring in step 2. His144 may also play a role in this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity of the proposed mechanism is discussed for other PQQ-containing dehydrogenases.

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.

Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli recombinant strain. The apoenzyme (lacking the cofactor pyrroloquinoline quinone, PQQ) was solubilized with Triton X-100 and purified to homogeneity. Reconstitution of the apoenzyme to full activity in the assay was achieved with a stoichiometric amount of PQQ in the presence of Mg2+. Just as for other PQQ-containing dehydrogenases where Ca2+ fulfills this role, Mg2+ anchors PQQ to the mGDH protein and activates the bound cofactor. This occurs in a precise way since high anomer specificity was found for the enzyme toward the sugars tested. Although the steady-state-type kinetics were as expected for a dye-linked dehydrogenase (ping-pong) and the PQQ in it was present in oxidized form, addition of glucose to the holoenzyme resulted in a very slow but continuous production of gluconolactone; i.e., the reaction did not stop after one turnover, with O2 apparently acting as an (albeit poor) electron acceptor by reoxidizing PQQH2 in the enzyme. The surprisingly low reactivity with glucose, in the absence of dye, as compared to the activity observed in the steady-state assay appeared to be due to formation of an anomalous enzyme form, mGDH. Formation of normal holoenzyme, mGDH, reducing added glucose immediately to gluconolactone (in one turnover), was achieved by treating mGDH with sulfite, by reconstituting apoenzyme with PQQ in the presence of sulfite, or by applying assay conditions to mGDH (addition of PMS/DCPIP). As compared to other quinoprotein dehydrogenases, mGDH appears to be unique with respect to the mode of PQQ-binding, as expressed by the special conditions for reconstitution and the absorption spectra of the bound cofactor, and the reactivity of the reduced enzyme toward O2. The primary cause for this seems not to be related to a different preference for the activating bivalent metal ion but to the special way of binding of PQQ to mGDH.

The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-Å resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176 -195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4 -356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 Å resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10°rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 Å. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed. (S)-Mandelate dehydrogenase (MDH)1 from Pseudomonas putida is the second component in the four-enzyme mandelateutilization pathway that converts (R)-mandelate to benzoate (1). The other enzymes are mandelate racemase, which catalyzes the first reaction in the pathway, and benzoylformate decarboxylase followed by benzaldehyde dehydrogenase which catalyze the third and fourth reactions, respectively. This pathway enables the organism to grow on mandelic acid which serves as the sole source of carbon and energy. MDH catalyzes the oxidation of (S)-mandelate to benzoylformate ( Fig. 1) (2). It belongs to a highly homologous family of (S)-␣-hydroxy acidoxidizing enzymes that are found in both eukaryotes and prokaryotes (3). Flavocytochrome b 2 (FCB2) from yeast (4), long chain hydroxy acid oxidase from mammals (5), lactate monooxygenase from bacteria (6), and glycolate oxidase (GOX) from spinach (7) are some examples of the widespread members of this enzyme family. A few members of this family are bound to the cytoplasmic membrane, including MDH (2) and L-lactate dehydrogenase from Escherichia coli, L-lactate dehydrogenase from Acinetobactor calcoaceticus (8) and L-pantoyl dehydrogenase from Nocardia asteroides (8, 9). MDH belongs to the monotopic class of integral membrane proteins (10). As a characteristic feature of this class of proteins, a small segment of MDH (about 40 or so residues) penetrates one side of the phospholipid bilayer.There is high sequence similarity (ϳ30 -45% identity) between the members of the ␣-hydr...

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