Biosci. Biotechnol. Biochem., 72 (1), 260–264, 2008 Communication The Occurrence of a Novel NADH Dehydrogenase, Distinct from the Old Yellow Enzyme, 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 oxygen 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-heme chloroperoxidase. The N-terminal enzymes 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 oxidoreductase 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.