Ethanol ^ Acetaldehyde
Quinohemoprotein alcohol dehydrogenase (ADH III) is localized on the outer surface of the cytoplasmic membrane of the acetic acid bacteria, Acetobacter and Gluconobacter. ADH III has the most important role in vinegar production in oxidizing ethanol to acetaldehyde. The acetaldehyde generated is immediately oxidized to acetate by aldehyde dehydrogenase located close to ADH III on the same cytoplasmic membrane. As mentioned elsewhere, most people still believe that ethanol oxidation during vinegar production must be catalyzed by a cytosolic NAD-dependent ADH, even though the discovery of ADH III was reported in 1960s. ADH III catalyzes alcohol oxidation under acidic conditions at pH 3-4, which is a favorable biological environment for vinegar production. It is distinct from the cytosolic NAD-dependent ADH that oxidizes alcohol under fairly alkaline conditions at pH 9-11. When acetic acid bacteria are growing on ethanol, the enzymic activity of the cytosolic NAD-dependent ADH is very weak and almost undetectable.
Among a huge number of PQQ-dependent dehydrogenases, ADH III has been purified and demonstrated to be a typical example of a membrane-bound dehydrogenase . It was the first membrane-bund enzyme to be crystallized (in 1982) . The purified ADH III shows heme c type absorption spectra with absorption maxima at 555, 523, and 418 nm in the reduced enzyme. A shift in the y-peak to 413 nm was observed for the oxidized enzyme. ADH III consists of three different subunits: subunit I (80 kDa), subunit II (50 kDa), and subunit III (20 kDa) (Fig. 1.3). Subunit I contains the catalytic site involving PQQ as the primary co-enzyme and one heme c, forming a superbarrel structure surrounded by eight propeller structures based on tryptophan .
ADH III has been shown to donate electrons to ubiquinone embedded in the membrane phospholipids, and then to the terminal oxidase. Although no ADH III subunits have a transmembrane domain, subunit II (cytochrome subunit) has been shown to have a ubiquinone reacting site and to have two amphiphilic a-helices as a possible membrane anchor . ADH III also seems to bind to the membrane and thus to transfer the reducing equivalent to ubiquinone via subunit
II. Subunit II involves three heme c components and binds the ADH III to the cytoplasmic membrane [6, 7]. The physiological function of subunit III is still unknown but it is obviously important in the ADH III reaction. If subunit III is deleted, ethanol oxidation no longer takes place.
The ethanol oxidase system is composed of three simple components: ADH III, ubiquinone-9 (UQ9) in the case of Acetobacter or ubiquinone-10 (UQ10) in Gluco-nobacter, and the transmembrane terminal oxidase, which functions as an ubi-quinol oxidase. The electron generated is transferred to UQ9 or UQ10 converting it to the reduced form, ubiquinol-9 or ubiquinol-10, which in turn are oxidized to UQ9 or UQ10 again. The terminal oxidase transfers the reducing equivalent across the cytoplasmic membrane, yielding a proton gradient (inside negative) allowing bioenergy generation. Thus, acetic acid bacteria are able to obtain bioenergy during substrate oxidation on the outer surface of the cytoplasmic membrane. This hypothesis has been confirmed using a reconstituted proteoliposome of the alcohol oxidase system involving ADH III, UQ9 or UQ10 and the terminal oxidase . A clear proton gradient across an artificial proteoliposome membrane vesicle is formed by the addition of substrate and can be followed by quenching of fluorescent dye along with alcohol oxidation. For more details, refer to [1, 5].
ADH III catalyzes the irreversible oxidation of primary alcohols except for methanol. Alcohol oxidation is conducted with different types of catalyst containing ADH III such as growing cells (vinegar production), resting cells, isolated membrane fractions, and purified ADH III. In this context, ADH III has been use in the construction of alcohol sensors or alcohol biosensors that are able to interact directly with an electrode. Unlike the NAD(P)-dependent alcohol dehydrogenases, alcohol oxidation is catalyzed under fairly acidic conditions at pH 3-6. Ethanol-grown cells of Acetobacter or Gluconobacter show a strong ethanol-oxidizing activity with the membrane fraction while a little enzyme activity of NAD(P)-dependent alcohol dehydrogenase is observed .
Ethanol oxidation with an NAD (P)-dependent alcohol dehydrogenase usually shows a pH optimum under highly alkaline conditions at pH 9-11 and aldehyde reduction to alcohol favorably occurs under acidic conditions at pH 5-6. On the other hand, if ADH III is deleted by means of genetic mutation, the enzyme activity of ADH III disappears in the membrane fraction and the cytosolic NAD(P)-dependent ADH becomes predominant and the specific activity increased remarkably. Acetate production with such a mutant no longer takes place and the total cell growth increases, because ethanol added to the culture medium is fully converted to carbon and energy sources for the mutant .
Among strains of acetic acid bacteria, acetate overoxidation (also known as acetate peroxidation) is an unfavorable phenomenon in acetate brewery. Our understanding of the molecular mechanism and physiological aspects of acetate peroxidation have progressed [6, 11]. Structural studies of ADH III by means of X-ray crystallography are now being undertaken.
In the case of ADH III of acetic acid bacteria, direct electron transfer to the electrode has been shown by direct binding of the enzyme to the hydrophobic surface of a gold electrode, which is able to transfer electrons directly to the electrode without any help of electron mediator [12, 13]. Such direct electron transfer to the electrode with ADH III has been confirmed in the case of the enzyme being entrapped onto a platinum electrode with polypyrrole . This intermolecular electron transfer reaction has also been observed in two quinohe-moproteins: ADH IIB (soluble quinohemoprotein ADH, see below) and ADH III. Based on the crystal structure of ADH IIB [15-17], it can be seen that the heme c domain in the C-terminus of the primary structure is distant from the N-terminal PQQ domain, which is highly homologous to the superbarrel structure of other PQQ-dependent enzymes , and covers the PQQ domain. The distance between the heme c and PQQ is about 20 A in the reduced form, which seems to be a little far for direct electron transfer from PQQ to heme c. Therefore, the electron transfer from PQQ to heme c could occur via an amino acid backbone present between both domains, or directly, upon a conformational change due to oxidation. The same kind of electron transfer may be expected in the quinohe-moprotein subunit of ADH III.
Furthermore, in the multimeric enzyme it has been suggested that electrons extracted from ethanol at the PQQ site could be transferred via the heme c site in the subunit I to one of three heme c sites in the cytochrome subunit (subunit II), and the electrons passed to ubiquinone through two of the heme c moieties (Fig. 1.3) [18, 19]. In the case of ADH III, subunit II has been shown to have a ubiquinone reacting site despite having no transmembrane domain. Thus, ubi-
quinone reduction in ADH III may occur via electron transfer from PQQ through three of the four heme c moieties present. The electron transfer kinetics of qui-noproteins or quinohemoproteins may become critical from the biotechnological point of view, especially when applying them to alcohol biosensors.
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