Introduction

Many membrane-bound dehydrogenases in the periplasmic space or on the outer surface of the cytoplasmic membrane of acetic acid bacteria and other aerobic

Gram-negative bacteria have been classified as PQQ- or FAD-dependent dehydrogenases [1]. Most of the enzymes are closely associated with oxidative fermentation in industry, catalyzing an incomplete one-step oxidation, allowing accumulation of an equivalent amount of corresponding oxidation products outside the cells. The active sites of individual enzymes face the periplasmic space as illustrated in Fig. 1.1.

All enzyme reactions are carried out by periplasmic oxidase systems, including alcohol- and sugar-oxidizing enzymes of the organisms. D-Glucose, ethanol, and many other substrates are oxidized by the dehydrogenases (shown as PQQ or FAD, except for aldehyde dehydrogenase) that are tightly bound to the outer surface of the cytoplasmic membranes of the organism. These membrane-bound enzymes irreversibly catalyze incomplete one-step oxidation and the corresponding oxidation products accumulate rapidly in the culture medium or reaction mixture. The electrons (e~) generated by the action of these dehydrogenases are transferred to ubiquinone in the membrane. The reducing equivalents are further transferred to the terminal ubiquinol oxidase in the cytoplasmic membranes. Thus, the organisms generate bioenergy through the enzyme activities of PQQ-and FAD-dependent dehydrogenases. The outer membrane of the organism forming the periplasmic space is omitted from Fig. 1.1. Many different NAD- or NADP-dependent dehydrogenases in the cytoplasm have no function in oxidative fermentation, and thus are not shown in Fig. 1.1.

Keto Fructose

Acetaldehyde 5-Keto-D-fructose

Fig. 1.1 Membrane-bound PQQ- and FAD-dependent primary dehydrogenases on the outer surface of acetic acid bacteria.

Acetaldehyde 5-Keto-D-fructose

Fig. 1.1 Membrane-bound PQQ- and FAD-dependent primary dehydrogenases on the outer surface of acetic acid bacteria.

At present, of the enzymes exploited as either PQQ-dependent or FAD-dependent dehydrogenases, aldehyde dehydrogenase is the only one that is known to use a molybdopterin coenzyme. Unlike the cytoplasmic oxidoreductas-es, no energy is required for substrate intake into the periplasm and pumping out the oxidation products across the outer membrane as shown in Fig. 1.2.

Microbial production of L-sorbose, aldehyde, and 2-keto-D-gluconate are the examples shown in Fig. 1.2. All substrates are oxidized by the respective membrane-bound dehydrogenase, of which active site faces the periplasmic space formed between the outer membrane and the cytoplasmic membrane. The dehy-drogenase then donates electrons to ubiquinone (UQ) that in turn transfers them to the terminal ubiquinol oxidase. The terminal oxidase generates an electrochemical proton gradient either by charge separation or by a proton pump or by both during substrate oxidation by the membrane-bound enzymes, allowing the organism to acquire bioenergy through substrate oxidation.

Traditionally, acetate fermentation (vinegar production) and L-sorbose fermentation are typical examples of oxidative fermentation and the classic case of microbial bioconversion. Our understanding of the mechanism of oxidative fermentation, however, was not elucidated until relatively recently. Most enzymes involved in oxidative fermentation are associated closely with industrial applications for useful biomaterial production. The production of acetate, L-sorbose, D-gluconate, dihydroxyacetone, and others developed as a practical industry before the clarification of the molecular mechanisms of the responsible enzymes. It was in 1970s that we started to clarify the molecular mechanisms of the individual enzymes involved in oxidative fermentation.

Before describing the actions of the individual PQQ- and FAD-dependent dehydrogenases, it is worth clarifying the common physiological roles and localizations of PQQ- and FAD-dependent dehydrogenases in acetic acid bacteria and

Fig. 1.2 Membrane-bound dehydrogenase-dependent periplasmic oxidase systems.

other microorganisms. Many people still believe that acetate is produced by the cytosolic NAD(P)-dependent alcohol dehydrogenase and keto-D-gluconate by the cytosolic NADP-dependent D-gluconate dehydrogenase located in the cytoplasm. Such a serious confusion is probably caused by the confused of localization of the enzymes concerned. Both membrane-bound enzymes and NAD(P)-dependent enzymes sometimes occur in the same cell-free extract when bacterial cells are broken down and the cell-free extract is prepared. Some periplasmic enzymes, such as quinoprotein methanol dehydrogenase in methylotrophs, are readily solu-bilized when the cell-free extract is prepared [2]. Given that oxidative fermentation is only functional under fairly acidic conditions, D-gluconate oxidation with an NADP-dependent enzyme observed at alkaline pH is unlikely to participate directly in keto-D-gluconate production under acidic conditions.

Although FAD is linked covalently to FAD-dependent enzymes and PQQ is tightly bound to enzyme proteins (though all PQQ-dependent enzymes (quino-proteins) contain PQQ as dissociable form), most of the membrane-bound dehydrogenases indicated earlier were stable and active without exogenous addition of the responsible coenzyme, giving the impression that they were coenzyme-independent or NAD (P)-independent dehydrogenases. However, when the cellfree extract is centrifuged under stronger centrifugal force (e.g. 68 000 x g for 60 min), the enzyme activity of the membrane-bound enzymes precipitates as the membrane fraction, while the majority of NAD (P)-dependent enzymes exist in the supernatant. A typical membrane-bound dehydrogenase can be freed from NAD (P)-dependent enzyme activity by such simple fractionation of cell-free extract. The membrane-bound dehydrogenases are only solubilized by the aid of detergents. Addition of a chaotropic agent like KCl sometimes increases the recovery of solubilized enzyme.

According to the physiological roles of membrane-bound and periplasmic enzymes, it should be beneficial to detoxify cellular toxic compounds such as methanol or amines outside cells (for example, in the periplasmic space) and not in the cytoplasm. When a cell-free extract is fractionated with ammonium sulfate, membrane-bound dehydrogenases tend to precipitate at relatively low concentrations of ammonium sulfate, in contrast to NAD(P)-dependent enzymes. Sometimes unexpectedly low enzyme recovery is seen after purification of cell-free extracts on ion exchange chromatographic column, suggesting the presence of membrane-bound enzymes.

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