Appl Microbiol Biotechnol (2010) 86:12571265 DOI 10.

1007/s00253-010-2529-z

MINI-REVIEW

Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology
Toshiharu Yakushi & Kazunobu Matsushita

Received: 25 January 2010 / Revised: 25 February 2010 / Accepted: 25 February 2010 / Published online: 20 March 2010 # Springer-Verlag 2010

Abstract Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) of acetic acid bacteria is a membrane-bound enzyme involved in the acetic acid fermentation by oxidizing ethanol to acetaldehyde coupling with reduction of membranous ubiquinone (Q), which is, in turn, re-oxidized by ubiquinol oxidase, reducing oxygen to water. PQQ-ADHs seem to have co-evolved with the organisms fitting to their own habitats. The enzyme consists of three subunits and has a pyrroloquinoline quinone, 4 heme c moieties, and a tightly bound Q as the electron transfer mediators. Biochemical, genetic, and electrochemical studies have revealed the unique properties of PQQADH since it was purified in 1978. The enzyme is unique to have ubiquinol oxidation activity in addition to Q reduction. This mini-review focuses on the molecular properties of PQQ-ADH, such as the roles of the subunits and the cofactors, particularly in intramolecular electron transport of the enzyme from ethanol to Q. Also, we summarize biotechnological applications of PQQ-ADH as to enantiospecific oxidations for production of the valuable chemicals and bioelectrocatalysis for sensors and fuel cells using indirect and direct electron transfer technologies and discuss unsolved issues and future prospects related to this elaborate enzyme. Keywords Alcohol dehydrogenase . Acetic acid bacteria . Pyrroquinoline quinone . Ubiquinone . Bioelectrocatalysis
T. Yakushi : K. Matsushita (*) Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi,, Yamaguchi, 753-8515, Japan e-mail: kazunobu@yamaguchi-u.ac.jp

Introduction Acetic acid bacteria are obligate aerobes and a group in the Alphaproteobacteria. More than 10 genera, including Acetobacter, Gluconobacter, and Gluconacetobacter, belong to acetic acid bacteria (Yamada and Yukphan 2008). Many of them have a characteristically active respiratory chain oxidizing several alcohols, sugars, and sugar alcohols to accumulate their corresponding oxidation products in the culture medium (see Matsushita et al. 1994 and Matsushita et al. 2004 for reviews). Acetic acid fermentation from ethanol, also known as vinegar production, by acetic acid bacteria is accomplished by two sequential catalytic reactions of membrane-bound, pyrroquinoline quinonedependent alcohol dehydrogenase (PQQ-ADH) and membrane-bound aldehyde dehydrogenase (Fig. 1). PQQ-ADH is a quinohemoproteincytochrome c complex bound to the periplasmic side of the cytoplasmic membrane and catalyzes the first step of acetic acid production to oxidize ethanol by transferring electrons to membranous ubiquinone (Q). The respiratory chain of acetic acid bacteria branches into the cytochrome bo3 ubiquinol oxidase and a cyanide-insensitive bypass oxidase at the Q site, both of which reduce oxygen to water. Thus, PQQ-ADH functions as the primary dehydrogenase in the ethanol oxidation respiratory chain. The complete genome sequence of Acetobacter pasteurianus, a vinegar producer, revealed a redundancy of genes encoding PQQ-ADH, although its physiological meanings are obscure (Azuma et al. 2009). We have characterized PQQ-ADH, which has a central role in vinegar production, in the molecular and catalytic properties for a long time. Here, we summarize the

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acetic acetethanol aldehyde acid

Appl Microbiol Biotechnol (2010) 86:12571265

2H+
PQQPQQADH ADH ALDH ALDH PQQADH

Periplasm CioAB

Cytochrome bo3

Cytoplasmic membrane

Cytoplasm

2H+ + 1/2O

KCN

H O 2H+

2H+ + 1/2O

H O

Fig. 1 An overview of the ethanol oxidation respiratory chain of acetic acid bacteria. Ethanol is oxidized to acetic acid by a sequential action of PQQ-ADH and membrane-bound aldehyde dehydrogenase (ALDH), reducing Q in the cytoplasmic membrane. Then the respiratory chain braches to energy-producing cytochrome bo3 ubiquinol oxidase (dark gray arrows) and less energy-producing

cyanide insensitive oxidase CioAB (light gray arrows), both of which reduce oxygen to water. PQQ-ADH also participates in a cyanideinsensitive respiratory chain, but its electron acceptor remains unknown. Thick double arrow indicates a possible interaction between PQQ-ADH and CioAB

molecular properties, mode of action, applications, and biotechnological perspectives of PQQ-ADH and discuss unsolved issues.

An overview of PQQ-ADH PQQ-ADHs were purified from different genera of acetic acid bacteria, Acetobacter, Gluconacetobacter, Gluconobacter, and Acidomonas as summarized in Table 1, and shown to consist of subunits I, II, and III, except for PQQADHs purified from Gluconacetobacter species, which consist of only the subunits I and II. The subunit I encoded
Table 1 Molecular organizations of PQQ-ADHs from various acetic acid bacteria

in the adhA gene is approx. 80 kDa in the molecular size and contains pyrroquinoline quinone (PQQ) and a heme c moiety as the prosthetic groups (Inoue et al. 1989). This subunit functions as the catalytic site for ethanol oxidation (Matsushita et al. 1996). The subunit II encoded in the adhB gene is approx. 50 kDa in the molecular size and contains three heme c moieties (Inoue et al. 1992; Tamaki et al. 1991) and presumably a Q, which is tightly bound to the protein (Matsushita et al. 2008). The bound Q seems to work as the redox mediator to the bulk Q, the physiological electron acceptor in the cytoplasmic membrane. Thus, the subunit II works as the electron mediator from the subunit I to the membranous Q.

Species

Strains

Molecular sizes of subunits (kDa) I II 72 72 74 76 76 76 72 72 72 71 71 85 80 50 44 44 55 55 55 44 45 45 44 44 49 54 III 15 20 16 16 16 16 14 8

References

A. lovaniensis A. pasteurianus

Formerly Acetobacter polyoxogenes

b

G. polyoxogenesa G. europaeus G. intermedius G. diazotrophicus G. xylinusb G. oxydans A. methanolica

IFO3284 NCI1452 KKP584 IFO3191 SKU1108 MSU10 NBI1028 V3 JK3 PAL5 IFO12528 JCM6891

(Matsushita et al. 1992) (Kondo et al. 1995) (Trcek et al. 2006) (Kanchanarach et al. 2009) (Kanchanarach et al. 2009) (Kanchanarach et al. 2009) (Tayama et al. 1989) (Trcek et al. 2006) (Trcek et al. 2006) (Gmez-Manzo et al. 2008) (Gmez-Manzo et al. 2008) (Adachi et al. 1978b) (Frbortov et al. 1997)

Gluconacetobacter xylinus

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PQQ-ADH complex can be partially dissociated into a sunbunit I/III complex and the subunit II at least in case of Gluconobacter oxydans (formerly Gluconobacter suboxydans) enzyme (Matsushita et al. 1996). The subunit I/III complex has ethanol:ferricyanide oxidoreductase activity, but does not have ethanol:Q-1 (an artificial Q analogue) oxidoreductase activity. On the other hand, the subunit II has no ADH activity. When the subunit I/III complex is reconstituted with the subunit II, the reconstituted PQQADH complex regains Q-1 reductase activity, as well as ferricyanide reductase activity. Taken together, it can be concluded that the subunit II is responsible for Q reduction. The subunit III encoded in the adhS gene is approx. 15 kDa in the molecular size and suggested to have no prosthetic groups related to redox reaction from biochemical experiments and its predicted amino acid sequences (Kondo et al. 1995). Indeed, PQQ-ADHs purified from Gluconacetobacter species do not contain the subunit III (Tayama et al. 1989).

(subunit I) mutant strain. It is reasonable that a ubiquinonereacting subunit, i.e., the subunit II, is responsible for binding to the membrane. It is plausible that the PQQ-ADH complex is completed in vivo by assembling the precomplexed subunits I and III to the subunit II located on the membrane surface because the isolated subunit II and subunit I/III complex are re-assembled into a PQQ-ADH complex by simple mixing and short incubation in vitro (Matsushita et al. 1996).

Redox potentials of four heme c sites: insights into the intramolecular electron transport PQQ-ADH has four hemes c as redox cofactor for ethanol oxidation, i.e., ferricyanide-oxidized PQQ-ADH can be reduced by addition of ethanol. Oxidation of ethanol takes place at the PQQ site that is a two-electron redox mediator, i.e., PQQ may be reduced to PQQH2 by ethanol oxidation. Then, electrons from PQQH2 would be transferred to multiple hemes c and a tightly bound Q. Finally, the enzyme reduces bulk Q, which is a two-electron redox molecule involved in intermolecular electron transport to the terminal ubiquinol oxidase. Our understanding of intramolecular electron transport of PQQ-ADH based on the redox titration study for the Acidomonas methanolica (formerly Acetobacter methanolicus) enzyme is summarized in Fig. 2 (Frbortov et al. 1998). Because PQQADHs including the A. methanolica enzyme have Q-1 reductase activity at acidic pH (pH 45), shown are only the values for redox potential of hemes c at pH 4.5, more physiological conditions than pH 7.0. Electrons of ethanol (redox potential at pH 4.5, EpH4.5: 80 mV) extracted at the
Sub III

Biogenesis of the PQQ-ADH complex The primary sequences of the AdhA (subunit I), AdhB (subunit II), and AdhS (subunit III) components suggest that these are all translated as precursor forms with the signal sequence for translocation across the cytoplasmic membrane by the Sec machinery (Pugsley 1993). Since the transit through the Sec machinery generally occurs in the unfolded state, proteins are folded into their programmed structures at the final destinations. The cofactors, PQQ as well as hemes c, are presumably assembled along protein folding in the periplasmic space. Q seems to bind to PQQADH even after protein folding because Q-free PQQ-ADH can bind a Q analog in two distinguishable kinetic manners, of which high affinity cannot be observed in Q-bound PQQADH (Matsushita et al. 2008). The subunit III seems to work as a molecular chaperone for folding and/or maturation of the subunit I, which is speculated from the following findings. The subunit III exists freely in the periplasmic space besides in the PQQ-ADH complex on the cytoplasmic membrane. Mutant strains defective in the adhS gene of A. pasteurianus lose ADH activity because they produce only the subunit II but fail to produce the subunit I as well as the subunit III (Kondo et al. 1995; Masud et al. 2010). The primary sequences of the mature forms of three subunits suggest that there is no hydrophobic segment responsible for membrane binding. Genetic experiments suggest that the subunit II, but not the subunits I and III, is a membrane binding subunit (Kondo et al. 1995). In the absence of the subunit II, the subunits I and III, which are presumably in a complex, are detected in a soluble fraction rather than a membrane fraction. However, the subunit II alone is detected in the membrane fraction of an adhA

Redox potential (pH 4.5)

ethanol
PQQ

acetaldehyde PQQ heme c I I eeSub I

mV

-17 24 187 190 255 220

eII1
Sub II

?
e-

heme c II1 heme c II2 heme c II3 Q

II2 eQ Q

II3 eQH2

Fig. 2 Model for the intramolecular electron transport of PQQ-ADH. Left panel: Electrons from ethanol are transported to PQQ and likely along the gray arrows to bound Q through multiple hemes c. On the other hand, electrons from ubiquinol are transported along the black arrows to the heme c II3 site and then to unknown electron acceptor shown by question marks. Light gray Q, bound Q; black Q, bulk Q. Right panel: Redox potentials at pH 4.5 for the redox cofactors in PQQ-ADH are shown in millivolts. The arrows indicate a proposed intramolecular electron transport path upon ethanol:Q oxidoreduction

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PQQ site (EpH4.5: 17 mV) are transferred to the heme c site (I, EpH4.5: +24 mV) in the subunit I, to one of the heme c site in the subunit II (II1, EpH4.5: +187 mV), which has the lowest redox potential in the subunit II. The electrons are transferred further to another heme c site (II2, EpH4.5: +190 mV), which has a middle redox potential, then finally to the bound Q (EpH4.5: +220 mV). By analogy to the cytochrome bo3 ubiquinol oxidase (Sato-Watanabe et al. 1994), the bound Q is presumably a site for binding of membranous bulk Q or ubiquinol (Matsushita et al. 2008). The subunit II has another heme c site (II3, EpH4.5: +255 mV) that is presumably involved in its ubiquinol oxidation activity (see below) rather than Q reduction because the redox potential of this heme c site is higher than that of Q.

Inactive form of PQQ-ADH has higher ubiquinol oxidation activity Acetic acid bacteria produce an inactive form of PQQADH, as well as the active enzyme under acidic or high aeration conditions (Matsushita et al. 1995). The inactive PQQ-ADH has 10-times lower Q-1 reductase activity, although there is no difference in the subunit composition or the prosthetic groups. The inactive enzyme can be converted in vitro to another form of PQQ-ADH, which is highly active but different from the active enzyme. Purified PQQ-ADH exhibits a ubiquinol:ferricyanide oxidoreductase activity besides ethanol:Q-1 and ethanol: ferricyanide oxidoreductase activities (Matsushita et al. 1999). It is noteworthy that ubiquinol:ferricyanide oxidoreductase activity is higher in the inactive PQQ-ADH than in the active enzyme. Evidence for ubiquinol oxidation activity of PQQ-ADH suggests that PQQ-ADH participates in a regulation for redox levels of ubiquinone/ubiquinol in the cytoplasmic membrane, even if the physiological electron acceptor for this reaction still remains unknown. Thus, the inactive PQQ-ADH is likely active for ubiquinol oxidation in vivo, and important for physiology of acetic acid bacteria grown under acidic and high aeration conditions.

Possible roles for the bound Q The bound Q can be found in many redox enzymes, such as the cytochrome bo3 ubiquinol oxidase (Sato-Watanabe et al. 1994), the PQQ-dependent glucose dehydrogenase (Elias et al. 2004), and the thiols:ubiquinone oxidoreductase DsbB of Escherichia coli (Bader et al. 2000). Although a physiological role of the bound Q is obscure, it is assumed that it works as a site for the Q- or ubiquinol-binding as described earlier (Matsushita et al. 2008). It is proposed that the bound Q facilitates intramolecular electron transfer in mitochondrial alternative NADH dehydrogenase of Saccharomyces cerevisiae (Yamashita et al. 2007). As shown in Fig. 3, considered an intramolecular electron transport of PQQ-ADH, electrons from the two-electron redox molecule (PQQ) should be transferred to the one-electron redox centers (heme c and bound Q), and then the electrons are transferred to another two-electron redox molecule (bulk Q). During electron transfer from the two- to one-electron redox molecules or cofactors, or vice versa, radical intermediates such as a PQQ radical and a ubisemiquinone radical are likely produced (Duine et al. 1984). It is suggested that the mammalian succinate dehydrogenase stabilizes ubisemiquinone during the Q reduction (Ruzicka et al. 1975). Yankovskaya et al. (2003) anticipated a possibility that a reacting site for the bulk Q of the E. coli succinate dehydrogenase can be a site producing reactive oxygen species, if a ubisemiquinone intermediate is not stabilized. Thus, the bound Q of PQQ-ADH likely ensures its intramolecular electron transport to prevent leakage of electrons, producing undesired compounds such as reactive oxygen species. Our hypothetical model for intramolecular electron transport in PQQ-ADH and the roles of the bound Q in ethanol oxidation is depicted in Fig. 3. We are currently trying to detect such radical species in the PQQ-ADH molecule in various redox states; at least PQQ radical was clearly detected (unpublished observations).

Involvement of PQQ-ADH in the alternative electron transport chain More than 20 years ago, we found a branched respiratory chain in Gluconobacter species, which comprises cyanidesensitive and cyanide-insensitive ones (Ameyama et al. 1987). Cyanide is a respiratory inhibitor that binds to hemecopper binuclear center (oxygen-binding site) of the terminal oxidases such as the cytochrome aa3 complex of mitochondoria and the cytochrome bo3 complex of E. coli. Cyanide naturally occurs as cyanogenic glycosides found in fruits, which can infer a reasonable speculation that acetic acid bacteria have developed an ability to respire even in the presence of cyanide because they are associated with fruits and flowers in nature. The major terminal oxidase in the oxidative fermentation of acetic acid bacteria is the cytochrome ba3 type ubiquinol oxidase (or bo3 depending on bacterial species), which is sensitive to cyanide. Purified PQQ-ADH, the cytochrome ba3 oxidase, phospholipids, and ubiquinone-9 can be reconstituted into proteoliposomes, artificial biological membranes that can lead generation of an inside-negative electrochemical potential coupling with ethanol oxidation (Matsushita et al. 1992). On the other hand, the cyanideresistant electron transport chain still remains largely unknown. Spontaneous G. oxydans mutants that lack the

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Ethanol oxidation

a
PQQ EtOH I II1 II2 Fe3+ Fe3+ Fe3+ Q Oxidized Fe3+ Fe3+ Fe3+ Q A PQQH2

PQQ EtOH Fe2+ Fe3+ Fe3+ Q B e- x 2

PQQH2 Fe2+ Fe2+ Fe3+ Q C

PQQ EtOH Fe2+ Fe2+ Fe2+ Q D

PQQH2 Fe2+ Fe2+ Fe2+ Q Reduced Q reduction

II2 Fe2+

c
Q e- x 2 Q QH2

II2 eQ Fe2+ eQ eQH2

Fig. 3 Hypothetical model for intramolecular electron transport of PQQ-ADH and possible role for bound Q. Alterations in redox states of PQQ, Fe ions of hemes c, and bound Q in PQQ-ADH are shown during ethanol oxidation (a). Oxidized state, and reduced or radical species are shown in black and light gray letters, respectively. Open arrows show a direction of electron flow. Parentheses indicate hypothetical intermediates. Intermediate A indicates an initial state of oxidized PQQ-ADH immediately after oxidation of a single ethanol molecule. When one electron of PQQH2 is transferred to heme c, intermediate A may be changed to intermediate B, they are

electrically equivalent to each other. By further oxidation of second molecule of ethanol, intermediate C that can be equivalent to D may be produced. Finally, the enzyme may be fully reduced by oxidation of another ethanol molecule. As for reduction of bulk Q, two rounds of single electron transfer to bulk Q generate bulk QH2 through the bound Q in a form of ubisemiquinone (b). Another possibility for electron flow to reduce bulk Q is that two single electrons are transferred from bound Q in a form of ubisemiquinone and ferrous heme c II2 (c)

subunit II of PQQ-ADH have relatively less cyanide resistance in their respiratory chain. When the subunit II is reconstituted in the membranes prepared from such the mutant strains, activity of the cyanide-resistant respiration can be elevated, as well as ethanol:ferricyanide oxidoreductase and ethanol oxidation activities (Matsushita et al. 1991). Furthermore, when the wild-type G. oxydans cells are grown under acidic conditions, the amounts of PQQADH, the subunit II in particular, are elevated parallel with the increase in the cyanide-resistant respiration (Matsushita et al. 1989; Shinagawa et al. 1989). These lines of evidence suggest that PQQ-ADH or the subunit II participates at least partially in the cyanide-resistant respiratory chain. We recently showed that genes for CioA/CioB (GOX278GOX279) are required for the cyanide-resistant respiratory chain of G. oxydans IFO12528 (Mogi et al. 2009). CioAB is related to the cytochrome bd ubiquinol oxidase but more homologous to CioAB, cyanide insensitive oxidase, of Pseudomonas aeruginosa (Cunningham et al. 1997). The IFO12528 cioA strain completely lost cyanide-resistant respiratory activity. Based on evidence that CioAB is thus essential for the cyanide-resistant respiratory chain, how do we explain the role of the ubiquinol oxidation activity of PQQ-ADH and the correlation of PQQ-ADH with cyanide-resistant respiration as described earlier? One possible explanation is that PQQADH mediates a function of CioAB, i.e., PQQ-ADH may interact with CioAB (Fig. 1). We currently try to find molecules that associate with PQQ-ADH in vivo, which

may help to reveal the role of PQQ-ADH in the cyanideresistant respiratory chain.

Broad substrate specificity of PQQ-ADH Substrate specificity of PQQ-ADH was reported for the G. oxydans and Acetobacter lovaniensis (formerly Acetobacter aceti) enzymes for the first time, where primary aliphatic alcohols having up to six carbons, except for methanol, are well oxidized by PQQ-ADH, but neither glycerol, formaldehyde, nor acetaldehyde are done. Later, however, acetaldehyde was reported as a substrate for PQQ-ADH (Matsushita et al. 1994). Acetaldehyde was also shown being oxidized by Gluconacetobacter diazotrophicus PQQADH at approx. 90% activity for ethanol (Gmez-Manzo et al. 2008). The A. pasteurianus enzymes also have acetaldehyde-oxidizing activity depending on the strains: PQQ-ADH from the MSU10 strain is 80% activity for ethanol, but the enzyme from SKU1108 is 20% activity for ethanol, respectively (Kanchanarach et al. 2009). Formaldehyde also is a substrate for PQQ-ADH (Shinagawa et al. 2006). Purified formaldehyde oxidizing enzyme also has ethanol:ferricyanide oxidoreductase activity and shows a similar subunit composition to that of PQQ-ADH. Finally, purified PQQ-ADH exhibits formaldehyde oxidizing activity when freshly prepared formaldehyde solution is used. More recently, it was reported that glycerol is oxidized by PQQ-ADH at the first carbon at high concentrations (>1 M)

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(Habe et al. 2009b). The activity is not saturated even at 2.2 M glycerol, which seems to be the limit for enzyme assay. The former work done at the concentrations of 100 mM might have underestimated glycerol-1 dehydrogenase activity of PQQ-ADH as approx. 1% activity for ethanol (Adachi et al. 1978a, b).

a
CH2OH CHOH CH2OH
glycerol PQQADH

H C HO

CH2OH CHO

L-glyceraldehyde

HO C H

CH2OH CHO

Enantiospecific oxidation of alcoholic compounds by PQQ-ADH Enantiometrically pure compounds are valuable as building blocks of pharmaceuticals and for other purposes. Enantiospecific oxidation of prochiral compounds and enantioselective oxidation of racemic compounds followed by kinetic resolutions are key technologies for production of enantiomers. Enantiospecific oxidation of alcoholic compounds by acetic acid bacteria was reported for the first time by Ohta et al. in 1982 (Ohta et al. 1982). The authors reported oxidation of prochiral compound 2-methylpropane-1,3diol to ()-(R)--hydroxyisobutylic acid with 83% ee by Gluconobacter roseus. Similar studies were reported as to enatiospecific oxidation of 2-phenyl-1-propanol (Molinari et al. 1999), 2-methylpropane-1,3-diol (Molinari et al. 2003), 2-butyl-1,3-propanediol (Mitsukura et al. 2007), and glycerol (Habe et al. 2009a) by acetic acid bacteria. Recently, collaboration of Habe et al. and our laboratory revealed through genetic and biochemical approaches that PQQ-ADH is primarily involved in the oxidation of glycerol to glyceraldehyde, presumably D-isomer, which is finally converted to D-glyceric acid (Fig. 4a) (Habe et al. 2009b). On the other hand, specific oxidation of an enantiomer by PQQ-ADH was also reported by Geerlof et al. (1994), i.e., PQQ-ADH specifically oxidizes the S enantiomer in racemic glycidol (2,3-epoxy-1-propanol, Fig. 4b). They also reported that such stereospecificity of PQQ-ADH in the membranes is higher than that of isolated enzyme. A key event for alteration in the selectivity occurs at the step for solubilization of PQQ-ADH from the membranes, although its molecular details are obscure (Machado et al. 1999).

D-glyceraldehyde

b
O H2C C CH2OH H
PQQADH

O H2C C

H CHO

(R)-glycidaldehyde

+
O H2C C CH2OH H

+
O H2C C CH2OH H

(R, S)-glycidol

(R)-glycidol

Fig. 4 Enantiospecific and enantioselective oxidations by PQQ-ADH. Enantiospecific oxidation of prochiral compound glycerol (a). PQQADH preferentially oxidizes glycerol to produce D-glyceraldehyde enclosed by a gray line. Enantioselective oxidation of (S)-glycidol (b). As enclosed by gray line, PQQ-ADH oxidizes selectively (S)-glycidol in racemic glycidol to produce (R)-glycidaldehyde

Applications of PQQ-ADH in bioelectrocatalyst for biosensors and biofuel cells Redox enzymes can be applicable in biosensor-based diagnosis, if they have high substrate specificity. Besides, such enzymes may also be applied in fuel cell technology for catalysis that is able to work at high concentrations of fuels. Particularly, NAD(P)+-independent oxidoreductases such as PQQ-ADH of acetic acid bacteria are expected for several applications because they do not require soluble cofactors. However, some small electromediator molecules

are generally required to couple the enzyme reaction to electrodes for electron currents. On the other hand, direct electron transfer (DET) between enzymes and electrodes, where the enzymes are directly attached to electrode surface such as graphite and metals, makes simpler devises possible and also minimizes energy loss during electron transfer (Ikeda and Kano 2003; Tkac et al. 2009). However, in that case, the enzymes should have additional redox centers such as hemes or [Fe-S] clusters apart from the active site for electron donor the substrate. Otherwise, ins and outs of electron donors may be prevented by attaching enzymes to electrodes. Two different approaches, indirect electron transfer method mediating by redox hydrogel polymer and DET method without any mediator, were reported to develop PQQ-ADH electrode. PQQ-ADH and an Os-based redox polymer, crosslinked polymer of 1-vinylimidazole with Os(4,4-dimethylbpy)2Cl, are immobilized on the surface of graphite or carbon-based screen printed (SP) electrode with poly (ethyleneglycol) diglycidyl ether (Niculescu et al. 2002). Osmium ions are critical factors for electron transfer from the enzyme to the electrode, because only less current can be recorded in the absence of the metal ions. Electric

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currents in the presence of ethanol depend on the voltage, and the maximum current could be observed at 300 mV against an Ag/AgCl electrode; the electric currents recorded at 300 mV with graphite electrode coated with PQQ-ADH linearly increase up to 1 mM and exhibit the KM value comparable to biochemical results (Niculescu et al. 2002). Thus, amperometric determination of ethanol is a potential application for the PQQ-ADH electrode. An on-line determination system for ethanol was constructed by using this PQQ-ADH electrode to ensure a development of wine fermentation (Niculescu et al. 2002). In laboratory-scale and winary-scale wine fermentation, ethanol concentrations can be traced in the course of fermentation by the PQQ-ADH electrode, where the data are comparable to conventional off-line enzymatic determination systems. Ikeda et al. (1993) reported that electricity is extracted from ethanol solution via electrodes such as gold, gold platinum, or graphite coated with PQQ-ADH in a DET manner. As schematically shown in Fig. 5, since PQQ-ADH seems to have separate binding sites for the electron donor ethanol and for the electron acceptor, hydrophobic Q, it can be an ideal DET enzyme. The currents are dependent on the ethanol concentrations at several hundred millivolts against an Ag/AgCl/saturated KCl electrode; determined KM values for ethanol are comparable to those from biochemical analysis. Thus, this electrode is also applicable to amperometric determination of ethanol. In these electrodes, maximum current (Imax) from ethanol depends on the electrode materials; the gold or goldplatinum electrode produces much higher Imax than the graphite electrode. Current efficiency for DET would be improved by attachment

efficiency of enzymes to electrodes and also by potential difference between redox centers of enzymes. As for the potential difference, PQQ-ADH has four hemes c having different redox potentials, but the subunit I has single heme c with 100 mV of redox potential at pH 7.0 in case of the G. oxydans enzyme, and thus, energy loss derived from intramolecular electron transfer is minimized, if the subunit I is applied to catalyst of DET. Thus, PQQ-ADH would be useful for biofuel systems. Recently, a more practical DET-based biofuel system was reported by combination of electrodes coated with FAD-dependent fructose dehydrogenase of Gluconobacter sp. as an anode and laccase of mushroom as a cathode (Kamitaka et al. 2007). Laccase accepts electrons from phenolic compounds to oxidize it making hydrogen peroxide as a byproduct, but in this case, laccase accepts electrons from the electrode rather than from the natural substrate. Using these electrodes, electricity can be extracted from fructose solution.

PQQ-ADH related to vinegar production Finally, we would like to discuss on the relation between PQQ-ADH and vinegar production. The genera of Acetobacter and Gluconacetobacter are important for vinegar production, and the former genus is involved in moderate concentration of acetic acid fermentation, while the latter in a high-concentration one. Gluconacetobacter europaeus V3 is highly tolerate to acetic acid: its high concentration of acetic acid fermentation can proceed from 3% ethanol in the presence of 7% acetic acid to 10% acetic acid in total (Trcek et al. 2006). Gluconacetobacter europaeus that accumulates high concentrations of acetic acid produces higher levels of PQQ-ADH in the membrane than A. pasteurianus that accumulates moderate levels of acetic acid. Not only the amount of the enzyme but also the properties are different from species to species. Gluconacetobacter europaeus PQQ-ADH is more resistant to acetic acid than the enzymes from A. pasteurianus and Gluconacetobacter intermedius, i.e., G. europaeus PQQ-ADH retains approx. 70% activity in the presence of 10% acetic acid, when compared to that under no acetic acid conditions. On the other hand, the A. pasteurianus and G. intermedius enzyme retain only 3% and 15% activity in the presence of 10% acetic acid, respectively. Resistance to acetic acid of G. europaeus PQQ-ADH reasonably relates to physiological conditions of this organism itself. We characterized PQQ-ADHs from several thermotolerant strains of A. pasteurianus (Kanchanarach et al. 2009). The SKU1108 and MSU10 strains isolated in Thailand are thermotolerant, and their PQQ-ADHs are relatively thermotolerant to that of the mesophilic A. pasteurianus strain. The

electrode

acetaldehyde ethanol

I II1 II2 Q II3

PQQ

Fig. 5 Schematic representation for PQQ-ADH electrode with DET. PQQ-ADH (light gray) attaches to electrode directly. Electrons from ethanol that are transported along the intramolecular pathway (solid arrows) may be transferred directly to electrode through multiple hemes c (dotted arrows), which function as built-in electromediators

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Appl Microbiol Biotechnol (2010) 86:12571265 Ameyama M, Matsushita K, Shinagawa E, Adachi O (1987) Sugaroxidizing respiratory chain of Gluconobacter suboxydans. Evidence for a branched respiratory chain and characterization of respiratory chain-linked cytochromes. Agric Biol Chem 51 (11):29432950 Azuma Y, Hosoyama A, Matsutani M, Furuya N, Horikawa H, Harada T, Hirakawa H, Kuhara S, Matsushita K, Fujita N, Shirai M (2009) Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus. Nucleic Acids Res 37(17):57685783 Bader MW, Xie T, Yu CA, Bardwell JC (2000) Disulfide bonds are generated by quinone reduction. J Biol Chem 275(34):26082 26088 Cunningham L, Pitt M, Williams HD (1997) The cioAB genes from Pseudomonas aeruginosa code for a novel cyanide-insensitive terminal oxidase related to the cytochrome bd quinol oxidases. Mol Microbiol 24(3):579591 Duine JA, Frank J, De Beer R (1984) An electron-nuclear doubleresonance study of methanol dehydrogenase and its coenzyme radical. Arch Biochem Biophys 233(2):708711 Elias MD, Nakamura S, Migita CT, Miyoshi H, Toyama H, Matsushita K, Adachi O, Yamada M (2004) Occurrence of a bound ubiquinone and its function in Escherichia coli membranebound quinoprotein glucose dehydrogenase. J Biol Chem 279 (4):30783083 Frbortov J, Matsushita K, Yakushi T, Toyama H, Adachi O (1997) Quinoprotein alcohol dehydrogenase of acetic acid bacteria: Kinetic study on the enzyme purified from Acetobacter methanolicus. Biosci Biotechnol Biochem 61(3):459465 Frbortov J, Matsushita K, Arata H, Adachi O (1998) Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study. Biochim Biophys Acta 1363(1):2434 Geerlof A, van Tol JBA, Jongejan JA, Duine JA (1994) Enantioselective conversions of the racemic C3-alcohol synthons, glycidol (2, 3epoxy-1-propanol), and solketal (2, 2-dimethyl-4-(hydroxymethyl)1, 3-dioxolane) by quinohaemoprotein alcohol dehydrogenases and bacteria containing such enzymes. Bioscience, Biotechnology and Biochemistry 42(1):815 Gmez-Manzo S, Contreras-Zentella M, Gonzlez-Valdez A, SosaTorres M, Arregun-Espinoza R, Escamilla-Marvn E (2008) The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus. Int J Food Microbiol 125(1):7178 Habe H, Fukuoka T, Kitamoto D, Sakaki K (2009a) Biotransformation of glycerol to D-glyceric acid by Acetobacter tropicalis. Appl Microbiol Biotechnol 81(6):10331039 Habe H, Shimada Y, Yakushi T, Hattori H, Ano Y, Fukuoka T, Kitamoto D, Itagaki M, Watanabe K, Yanagishita H, Matsushita K, Sakaki K (2009b) Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol. Appl Environ Microbiol 75(24):77607766 Ikeda T, Kano K (2003) Bioelectrocatalysis-based application of quinoproteins and quinoprotein-containing bacterial cells in biosensors and biofuel cells. Biochim Biophys Acta 1647(1 2):121126 Ikeda T, Kobayashi D, Matsushita F (1993) Bioelectrocatalysis at electrodes coated with alcohol dehydrogenase, a quinohemoprotein with heme c serving as a built-in mediator. J Electroanal Chem 361:221228 Inoue T, Sunagawa M, Mori A, Imai C, Fukuda M, Takagi M, Yano K (1989) Cloning and sequencing of the gene encoding the 72kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J Bacteriol 171(6):31153122 Inoue T, Sunagawa M, Mori A, Imai C, Fukuda M, Takagi M, Yano K (1992) Cloning and sequencing of the gene encoding the 45kilodalton subunit of alcohol dehydrogenase from Acetobacter aceti. J Ferment Bioeng 73(6):419424

activities of thermotolerant PQQ-ADHs are more slowly decreased under 55C treatment than that of mesophilic enzyme. Thus, it is reasonably speculated that PQQ-ADH of A. pasteurianus has been rapidly evolved fitting to its own habitat even in an intraspecies manner.

Prospects for future applications of PQQ-ADH Not only for the sensor and fuel cell technologies, enzyme electrode is an attractive form for applications of PQQ-ADH. As described earlier, PQQ-ADH has broad substrate specificity, but the reaction is regio- and enantiospecific. Purified PQQ-ADH can be assumed for catalysis in regio- and enantiospecific oxidation of hydroxyl group of artificial compounds in fine chemical industries, such as building blocks for pharmaceuticals. However, in case of free enzyme solution systems, such processes must be annoyed by separation of an oxidation product from some chemical oxidants, such as ferricyanide needed for enzyme turnover. On the other hand, enzyme electrode is an attractive catalyst for oxidation reaction because enzymes bound to electrode can be re-oxidized by electric current. In another case for using living bacteria, oxygen supply is critical for oxidation reactions and, thus, would be a bottleneck for large-scale productions. The roles of the subunits and intramolecular electron transport of PQQ-ADH have been uncovered step by step, although many specific issues still remain to be solved. Xray crystal structure of PQQ-ADH has been examined but not reported yet, which will provide us its structural information in the atomic levels. The structural studies may help to understand the active site structure related to enantioselectivity and broad substrate specificity of PQQADH. Such data also stimulate to understand intramolecular electron transport pathways, which may help to apply the enzyme to develop a more practical DET biocatalyst. Elucidation of action for PQQ-ADH catalysis by basic studies, including biochemical, molecular, spectral, and structural analyses, would help a development of enzymatic electrocatalysis for oxidation reactions in production of valuable fine chemicals.

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