Biosci. Biotechnol. Biochem., 72 (1), 260–264, 2008 Communication The Occurrence of a Novel NADH Dehydrogenase, Distinct from the Old Yellow , in Gluconobacter Strains

y Emiko SHINAGAWA,1; Yoshitaka ANO,2 Osao ADACHI,2 and Kazunobu MATSUSHITA2

1Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Ube 755-8555, Japan 2Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan

Received October 12, 2007; Accepted November 27, 2007; Online Publication, January 7, 2008 [doi:10.1271/bbb.70657]

A novel NADH dehydrogenase (NADH-dh) involving 3244 grown on a culture medium consisting of 20 g of FAD as coenzyme, distinct from NADPH dehydrogenase sodium D-gluconate, 5 g of D-glucose, 3 g of glycerol, (NADPH-dh, old yellow enzyme, EC 1.6.99.1), was 3 g of yeast extract, 2 g of polypeptone, and 200 ml of found in the same cytoplasmic fraction of Gluconobacter potato extract in 1 liter of tap water. NADH-dh and strains. Conventional artificial electron acceptors were NADPH-dh were assayed by reading the decreasing more effective than molecular in the NADH-dh absorbance of NADH and NADPH at 340 nm, by reaction. NADH-dh did not appear to be identical with essentially the same method as reported previously.2) any previously described flavoproteins, although the N- After the cell-free extract was applied to chromatog- terminal amino acid sequence showed 100% similarity raphy on a DEAE-Sephadex A-50 column, the two with a non- chloroperoxidase. The N-terminal came out at different positions. NADH-dh was amino acid sequence of NADPH-dh matched 100% a eluted from the column with 10 mM potassium phos- putative containing the old yellow en- phate buffer involving 5 mM -mercaptoethanol (KPB), zyme-like FMN-binding domain. NADH-dh might func- pH 7.2, containing 100 mM KCl. On the other hand, tion to regenerate NAD coupling with NAD-dependent NADPH-dh came out with 10 mM KPB, pH 7.2, con- dehydrogenases in the cytoplasm of Gluconobacter taining 250 mM KCl, under similar conditions, as strains. reported previously.2) Thus the two enzymes were separated from each other by simple column chroma- Key words: acetic acid bacteria; Gluconobacter strains; tography. NADH dehydrogenase; NADPH dehydro- NADH-dh was precipitated with ammonium sulfate at genase 65% saturation (43 g/100 ml), the pH was adjusted to 7.2 with ammonia, and the precipitate was dissolved in a NADPH dehydrogenase (NADPH-dh, old yellow en- minimum volume of KPB. NADH-dh was crystallized, zyme, EC 1.6.99.1) from yeast1) and Gluconobacter giving beautiful prisms, as shown in Fig. 1. High suboxydans2) catalyze NADPH oxidation to regenerate homogeneity was confirmed with crystalline NADH-dh NADP coupling with NADP-dependent dehydrogenases. as judged by analytical ultracentrifugation (Fig. 2) and Microbial NADPH-dh is a typical flavoprotein contain- native polyacrylamide gel electrophoresis (native- ing FMN as the coenzyme, while plant NADPH di- PAGE) (Fig. 2). The apparent sedimentation coefficient aphorase contains FAD and the plant enzyme does not was determined to be 4.1s. Molecular weight measure- link to oxygen as the electron acceptor.3) Most of these ment of NADH-dh by gel filtration gave 120,000. When are specific to NADPH in vivo, although NADH is developed in SDS-PAGE, NADH-dh gave only one oxidized in vitro to some degree of NADPH oxidation. band, corresponding to 30 kDa, implying that it was Although the physiological roles of NADPH-dh remain composed of four identical subunits (Fig. 2). The to be elucidated, cyclic regeneration of NADP is a prob- absorption spectra of NADH-dh showed two absorption able physiological role of NADPH-dh, as was explained maxima in the visible region, at 445 and 340 nm, with previously.1,2) clear shoulders at 468 and 420 nm (Fig. 3). NADH-dh In a series of studies on oxidative fermentation by appeared to be different from those flavoproteins acetic acid bacteria, NADH-dh was found in the same classified in EC 1.1.99.24–6) and EC 1.6.99.3,7,8) most cytoplasmic fraction of Gluconobacter oxydans IFO of which are unable to use oxygen as the electron

y To whom correspondence should be addressed. Tel: +81-836-35-6839; Fax: +81-836-21-7117; E-mail: [email protected] Abbreviations: NADH-dh, novel NADH dehydrogenase; PAGE, native polyacrylamide gel electrophoresis; NADPH-dh, NADPH dehydrogenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis A Novel NADH Dehydrogenase in Gluconobacter Strains 261 using oxygen as electron acceptor, as similarly found in the case of NADPH-dh.1,2) FAD was identified as the coenzyme by treating NADH-dh with acidic ammonium sulfate, partially converting NADH-dh to apo-enzyme, according to the method reported previously.9) The resulting partially resolved enzyme accompanied de- creased absorbance in the absorption spectrum in the visible region and decreased enzyme activity. The enzyme activity and also absorption spectrum were restored to the original level with the external addition of FAD, but not with FMN. Quantitative measurement of FAD in NADH-dh remains to be examined. As discussed previously,2) if NADPH-dh has a function regenerating NADP coupling with cytoplasmic NADP- dependent dehydrogenases, NADH-dh might function to regenerate NAD by coupling with NAD-dependent dehydrogenases in the cytoplasm of Gluconobacter Fig. 1. Photopicture of Crystalline NADH-dh from G. oxydans IFO strains, like ribitol dehydrogenase, D-sorbitol dehydro- 3244. genase, meso-erythritol dehydrogenase, cyclic alcohol dehydrogenase, and D-arabitol dehydrogenase.10) Isolation, purification, and crystallization of NADPH- acceptor. Molecular oxygen was available as an electron dh were conducted according to methods similar to acceptor in the NADH-dh reaction (giving 0.066 units/ those reported for the enzyme from G. suboxydans IFO mg), although p-benzoquinone (1.7 units/mg) and 2,6- 12528.2) Yellow-colored fractions eluted with a buffer dichlorophenolindophenol (9.3 units/mg) were more containing 250 mM KCl from DEAE-Sephadex A-50 effective than molecular oxygen. The characteristic was precipitated with ammonium sulfate at 65% absorption spectrum was reduced by the addition of saturation (43 g/100 ml), the pH was adjusted to 7.2 NADH, and the spectrum of NADH-reduced NADH-dh with ammonia, and the precipitate was dissolved in a was restored to the original level after the enzyme minimum volume of KPB. NADPH-dh was crystallized solution was bubbled with oxygen. Thus NADH as thin needles, as shown in Fig. 4. High homogeneity oxidation proceeded by NADH-dh in a cyclic manner was confirmed, as judged by analytical ultracentrifuga-

A B

kDa

20 min 107 94

35 min 52

37

50 min 28

65 min 19

Fig. 2. Sedimentation Patterns, Native-PAGE, and SDS-PAGE of NADH-dh. Sedimentation patterns were taken at 10 mg/ml in KPB at 20 C. Pictures were taken at 15-min intervals as indicated after reaching 60,000 rpm. A, Native-PAGE, 50 mg of NADH-dh was loaded to a disc gel. B, SDS-PAGE, 5 mg of the NADH-dh was loaded. The pre-stained standard marker proteins (Bio-Rad, Hercules, CA) were used with the following molecular sizes: 107 kDa, phosphorylase B; 94 kDa, bovine serum albumin; 52 kDa, ovalbumin; 37 kDa, carbonic anhydrase; 28 kDa, soybean trypsin inhibitor; 19 kDa, lysozyme. 262 E. SHINAGAWA et al.

2

1

Fig. 4. Photopicture of Crystalline NADPH-dh from G. oxydans IFO 3244.

0.1 3 ously,1,2) The NADPH-dh reaction proceeded in a cyclic manner using molecular oxygen as the electron acceptor, also similarly to the case of NADH-dh, as mentioned above. NADH-dh and NADPH-dh showed pH optima at 7-8 200 300 400 500 600 and 5.0 for NADH oxidation and NADPH oxidation, respectively. Different species of coenzyme, FAD and Wavelength (nm) FMN, were involved in NADH-dh and NADPH-dh, respectively, although the reactions catalyzed were Fig. 3. Absorption Spectra of NADH-dh. Line 1, NADH-dh (6.9 mg/ml) was taken in the visible region. similar to each other. The reaction mixtures (1 ml) Line 2, the absorption spectrum was taken after NADH-dh (line 1) contained 0.125 mmol of NADH and NADPH in McIl- was diluted to 1 mg/ml. Line 3, 2.5 mlof50mM NADH (0.125 mmol) vaine buffer, pH 8 and pH 5, in the NADH-dh reaction was added to the solution of line 2. and the NADPH-dh reaction respectively. One enzyme unit was defined as the amount of 1 mmol of NADH or NADPH oxidized per min using molecular oxygen as tion (Fig. 5), although the sedimentation pattern did not the electron acceptor. The oxidation rate of NADH to show a symmetric peak, suggesting that some smaller NADPH with NADH-dh gave 3.0, and that of NADPH components existed. Native-PAGE gave a single protein to NADH with NADPH-dh gave 2.0. Since the absolute band (Fig. 5). The apparent sedimentation coefficient measurements of their intrinsic the protein concentration 1% was determined to be 2.5s, corresponding roughly to a to the absorbance at 280 nm, Ecm. 280 nm, were not molecular weight comparable to 50,000–60,000. When determined, protein concentration was measured tenta- developed in SDS-PAGE, NADPH-dh gave two protein tively by a modified method of Lowry.11) The reaction bands, corresponding to 50 kDa and 15 kDa (Fig. 5). The rate by NADH-dh and also NADPH-dh was relatively 15-kDa band suggests that the slower sedimenting low when molecular oxygen was used as the electron components indicate impurities and distinctness from acceptor, as described above. As has been suggested as NADPH-dh. The absorption spectra of NADPH-dh were to the roles of NADPH-dh by Kataoka et al.,12,13) if examined, as shown in Fig. 6. NADPH-dh showed two different substrates to be reduced by NADH-dh and absorption maxima in the visible region, at 470 and NADPH-dh occur in the cytoplasm of organisms, if their 377 nm, with slight shoulders at 490 and 450 nm. The reaction rates are increased, their intrinsic substrates and absorption spectrum was reduced by the addition of their true physiological roles will finally be clarified. NADPH, and the spectrum of NADPH-reduced enzyme The 30-kDa protein of NADH-dh and the 50-kDa was restored to the original level after the enzyme protein of NADPH-dh showed N-terminal amino acid solution was bubbled with oxygen. p-Benzoquinone sequences PYITATDGTS and PTLFDPIDF respectively (0.75 units/mg), 2,6-dichlorophenolindophenol (3.04 when analyzed under the conditions as described units/mg), and cinnamaldehyde (13.89 units/mg) were previously.14) The detected amino acid sequence of more effective electron acceptors than molecular oxygen NADH-dh was 100% matching the sequence encoded by (0.054 units/mg) for NADPH-dh. As discussed previ- the locus GOX1766 in G. oxydans 621H genome, which A Novel NADH Dehydrogenase in Gluconobacter Strains 263 AB

kDa

15 min 107 94

30 min 52

37 45 min 28

60 min 19

Fig. 5. Sedimentation Patterns, Native-PAGE, and SDS-PAGE of NADPH-dh. Sedimentation patterns were taken at 10 mg/ml in KPB at 20 C. Pictures were taken at 15-min intervals as indicated after reaching 60,000 rpm. A, Native-PAGE, 50 mg of enzyme protein was loaded to a disc gel. B, SDS-PAGE, 5 mg of enzyme protein was loaded. The pre- stained standard marker proteins used were the same as in Fig. 2.

is annotated as a non-heme chloroperoxidase (alpha/ beta superfamily) (EC 1.11.1.10). The se- quence of NADPH-dh also showed 100% correspond- ence with the sequence encoded by GOX0502, annotated as a putative oxidoreductase containing the old yellow enzyme-like FMN-binding domain. As judged from previous data,2) NADPH-dh has the same function as the 2 enzyme in yeast. However, regarding the function NADH-dh as judged by N-terminal amino acid analysis, there is still some difficulty in accepting NADH-dh as a non-heme chloroperoxidase, due to many discrepant points in catalytic properties as well as in absorption spectra.15) The same observations described above were made in the cytoplasmic fraction of another Gluconobacter 1 strain, G. suboxydans IFO 3255, grown in a culture medium containing 1% glycerol, 0.3% yeast extract, 3 0.3% polypeptone, and 2 mM of CaCl2 (unpublished observations, Shinagawa). NADH-dh and NADPH-dh

0.1 might be ubiquitous enzymes in the cytoplasm of Gluconobacter strains, and the two enzymes must have important roles in cytoplasmic metabolism during oxidative fermentation. This report is perhaps the first indicating the occurrence of NADH-dh, as distinct from 200 300 400 500 600 NADPH-dh, and the two enzymes were different each other in biochemical significance. Wavelength (nm) Acknowledgments Fig. 6. Absorption Spectra of NADPH-dh. Line 1, NADPH-dh (9.2 mg/ml) was taken in the visible region. Line 2, the absorption spectrum was taken after the enzyme (line 1) Photopictures of crystalline NADH-dh and NADPH- was diluted to 1 mg/ml. Line 3, 2.5 mlof50mM NADPH dh were kindly made by Professor S. Tanaka of the (0.125 mmol) was added to the solution of line 2. Department of Biological and Environmental Sciences, 264 E. SHINAGAWA et al. Faculty of Agriculture, Yamaguchi University. Our pyridine nucleotide dehydrogenase of bovine erythro- sincere thanks go also to Dr. S. Moonmangmee of the cytes. Biochim. Biophys. Acta, 268, 629–637 (1972). Thailand Institute of Scientific and Technological 8) Hochstein, L. I., and Dalton, B. P., Studies of a Research for his kind assistance in analytical ultra- halophilic NADH dehydrogenase: purification and prop- centrifugation. Part of this work was supported by erties of the enzyme. Biochim. Biophys. Acta, 302, 216– 228 (1973). Grants-in-Aid for Scientific Research from JSPS (C 9) Yamada, H., Adachi, O., and Ogata, K., Putrescine 18580084, to E.S.). oxidase, a diamine oxidase requiring flavin adenine dinucleotide. Agric. Biol. Chem., 29, 1148–1149 (1965). 10) Adachi, O., Ano, Y., Toyama, H., and Matsushita, K., References Biooxidation with PQQ- and FAD-dependent dehydro- genases. In ‘‘Modern Biooxidation. Enzymes, Reactions 1) Akesson, A., Ehrenberg, A., and Theorell, H., Old and Applications,’’ eds. Schmid, R. D., and Urlacher, V. yellow enzyme. In ‘‘The Enzymes’’ 2nd ed. Vol. 7, eds. B., Wiley-VCH, pp. 1–41 (2007). Boyer, P. D., Lardy, H., and Myrback, K., Academic 11) Dully, J. R., and Grieve, P. A., A simple technique for Press, New York, pp. 477–494 (1963). eliminating interference by detergents in the Lowry 2) Adachi, O., Matsushita, K., Shinagawa, E., and method of protein determination. Anal. Biochem., 64, Ameyama, M., Occurrence of old yellow enzyme in 136–141 (1975). Gluconobacter suboxydans, and the cyclic regeneration 12) Kataoka, M., Kotaka, A., Hasegawa, A., Wada, M., of NADP. J. Biochem., 86, 699–709 (1979). Yoshizumi, A., Nakamori, S., and Shimizu, S., Old 3) Jagendorf, A. T., Chloroplast TPNH diaphorse. Methods yellow enzyme from Candida macedoniensis catalyzes Enzymol., 6, 430–434 (1963). the stereospecific reduction of the C=C bond of 4) Liao, S., Dulaney, J. T., and Williams-Ashman, H. G., ketoisophorone. Biosci. Biotechnol. Biochem., 66, Purification and properties of a flavoprotein catalyzing 2651–2657 (2002). the oxidation of reduced ribosyl nicotinamide. J. Biol. 13) Kataoka, M., Kotaka, A., Thiwthong, R., Wada, M., Chem., 237, 2981–2987 (1962). Nakamori, S., and Shimizu, S., Cloning and overexpres- 5) Zhao, Q., Yang, X. L., Holtzclaw, W. D., and Talalay, sion of the old yellow enzyme gene of Candida P., Unexpected genetic and structural relationships of a macedoniensis, and its application to the production of long-forgotten flavoenzyme to NAD(P)H:quinone reduc- a chiral compound. J. Biotechnol., 114, 1–9 (2004). tase (DT-diaphorase). Proc. Natl. Acad. Sci. USA, 94, 14) Saichana, I., Ano, Y., Adachi, O., Matsushita, K., and 1669–1674 (1997). Toyama, H., Preparation of enzymes required for 6) Wu, K., Knox, R., Sun, X. Z., Joseph, P., Jaiswal, A. K., enzymatic quantification of 5-keto-D-gluconate and 2- Zhang, D., Deng, P. S., and Chen, S., Catalytic proper- keto-D-gluconate. Biosci. Biotechnol. Biochem., 71, ties of NAD(P)H quinone -2 (NQO2), a 2478–2486 (2007). dihydronicotinamide riboside dependent oxidoreductase. 15) Otto, K., Hofstetter, K., Rothlisberger, M., Witholt, B., Arch. Biochem. Biophys., 347, 221–228 (1997). and Schmid, A., Biochemical characterization of StyAB 7) Adachi, K., and Okuyama, T., Study on the reduced from Pseudomonas sp. strain VLB120 as a two-compo- pyridine nucleotide dehydrogenase of bovine erythro- nent flavin-diffusible monooxygenase. J. Bacteriol., 186, cytes. I. Crystallization and properties of the reduced 5292–5302 (2004).