NADH dehydrogenase in Corynebacterium glutamicum

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1 NADH dehydrogenase in Corynebacterium glutamicum Nawarat Nantapong 1, Hirohide Toyama, Kazunobu Matsushita Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University ( 1 Present address: Veterinary Technology College, Kasetsart University) Abstract Type II NADH dehydrogenase (NDH-2) of Corynebacterium glutamicum was studied by preparing two mutant strains of NDH-2 disruption and over-expression. The effect of NDH-2 mutations on respiration-related metabolism was compared in both strains in order to determine the main role of this enzyme on the central metabolism. This study revealed that the coupling reaction of cytoplasmic and respiratory chain-linked dehydrogenases in malate and L-lactate oxidation systems, especially L-lactate oxidation system, functions as the NADH re-oxidation system when NDH-2 function was defected. Furthermore, NDH-2 was purified and characterized from the membrane of its overexpressed strain. Purified NDH-2 contains non-covalently bound FAD as a cofactor, and has an ability to catalyze electron transfer from NADH and NADPH to menaquinone and oxygen, respectively. The NADPH oxidation was also found to occur concomitant with the generation of superoxide anion. Introduction Idustrial production of glutamic acid as well as amino acids of the aspartic acid family from carbohydrates is carried out by a group of bacteria represented by Corynebacterium glutamicum. C. glutamicum is gram-positive, non-pathogenic, non-motile and non-sporulating ellipsoidal spheres or short rods. This bacterium requires biotin together with some addition of thiamine (vitamin B 1 ) for the growth. C. glutamicum is an aerobic bacterium using oxygen as terminal electron acceptor for the respiration. The respiratory chain includes at least three cytochrome complexes, cytochrome bc 1 complex, cytochrome aa 3 oxidase and cytochrome bd oxidase, which may couple electron transfer to the generation of an electrochemical proton gradient across the cytoplasmic membrane. Recent molecular and biochemical analyses together with information obtained from the genome sequence have shown that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features (Bott & Niebisch,

2 2003). Reducing equivalents obtained by the oxidation of various substrates are transferred to manaquinone via at least eight different dehydrogenases which are NADH dehydrogenase (NDH- 2), succinate dehydrogenase (SDH), malate:quinone oxidoreductase (MQO), pyruvate:quinone oxidoreductase, D-lactate dehydrogenase (D-LDH), L-lactate dehydrogenase (L-LDH), glycerol- 3-phosphate dehydrogenase and L-proline dehydrogenase, all containing flavin as the cofactor. From menaquinol, the electrons are passed either via cytochrome bc 1 complex to aa 3 type cytochrome c oxidase, or to the cytochrome bd type menaquinol oxidase. The former branch is exceptional, in that it does transfer electrons via two heme c moieties in cytochrome c 1 directly to the Cu A center in subunit II of cytochrome aa 3. The bc 1 complex and cytochrome aa 3 oxidase seems to form a super complex in C. glutamicum. The phenotype of defined mutants revealed that the bc 1 -aa 3 branch is of major importance for the energy generation. Changes of the efficiency of the oxidative phosphorylation caused by qualitative change of the respiratory chain or by a defective F 1 F 0 -ATP synthase have been shown to have strong effects on metabolism and amino acid production. Thus, the respiratory chain and the energy producing mechanism are an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering. NADH dehydrogenase is the main entry site of reducing equivalents from the central metabolism to the respiratory chain of all organisms having an aerobic or anaerobic electrontransport system. NADH dehydrogenase found in bacterial respiratory chains can be divided into three different types, type I NADH dehydrogenase (NDH-1), type II NADH dehydrogenase (NDH-2), and Na + -translocating NADH:quinone oxidoreductase. NDH-1, which is homologous to mitochondrial complex I, is composed of different subunits and has FMN and several iron-sulfur clusters as the prosthetic groups. This enzyme is able to pump protons from the cytosolic side to the periplasmic side. NDH-2 is a single subunit enzyme and bears flavin but no iron-sulfur clusters. Although the oxidation of NADH is extensively carried out by complex I in mammals, mitochondria from fungi contain an alternative NADH dehydrogenase, NDH-2, together with complex I in Neurospora crassa or without complex I in Saccharomyces cerevisiae. Similar to fungi, the bacterial respiratory chain has NDH-1 and NDH-2, or either one of them. Escherichia coli has NDH-1 and NDH-2 (Matsushita et al., 1987), which are encoded by the nuo operon and ndh gene, respectively. However, Paracoccus denitrificans has only NDH-1 and

3 Bacillus subtilis has only NDH-2. Although bacterial NDH-1 from E. coli and P. denitrificans has been well-characterized, NDH-2 has not been well studied except for E. coli NDH-2. The NADH dehydrogenase of C. glutamicum is NDH-2, which is able to partly work as an NADPH oxidase (Matsushita et al., 2001; Nantapong et al., 2004) and generate superoxide anion. Thus, NDH-2 of C. glutamicum has unique character compared with NDH-2 from other bacterial species. 1. Role of C. glutamicum NADH dehydrogenase on respiratory chain and metabolism The function of C. glutamicum NDH-2 (encoded in ndh gene) has been studied with ndh disrupted and over-expressed strains (Nantapong et al., 2004; 2005). Both NADH and NADPH oxidase activities could not be detected in the ndh disruptant strain, while in the over-expressed strain, the oxidation of NADH and NADPH in the membrane fraction was significantly higher than wild type strain KY9714. In glucose minimum medium, the growth was much lower in the over-expressed strain than the wild type and disruptant strains both of which had similar growth, suggesting that energy generation of the over-expressed strain might be lower than the others. However, the H + /O ratio was not much different among the strains. Since the over-expressed stain produced a higher level of O - 2, which depended on NADH or NADPH respiration, the delay in growth for this strain might be due to the generation of O - 2 although it seems that the overproduction of such a membrane-bound protein as NDH-2 may also have some negative effect on growth. Whereas, the growth of these strains on lactate minimum medium was something different, which seemed to correspond to the changes of L-LDH and L-lactate oxidase activities. The ndh disruptant strain caused the improvement of the growth rate and also exhibited a higher L-lactate-dependent enzyme activities than KY9714 did, while the decreasing of these activities and the much lower growth on lactate minimum medium was observed with the over-expressed strain. In the lactate minimum medium, since lactate, the carbon and energy source, can not be efficiently converted to pyruvate, an important precursor for TCA cycle, the ndh over-expressed strain is expected not to produce enough energy and cell materials requiring for the cell growth. The disruption and over-expression of NDH-2 did not affect very much on the components and also the contents of the respiratory chain. The findings could be supported by a study by Molenaar et al. (2000), where ndh disruption did not lead any severe growth defects. They observed that a growth defect could be seen only after double mutation with MQO,

4 suggesting that coupling between MQO and malate dehydrogenase (MDH) may be required for NADH re-oxidation instead of NDH-2. However, even the double mutant strain can grow, albeit slowly, in glucose minimum medium. As previously described, in the disruptant lacking NDH-2- dependent NADH oxidation, membrane-bound L-LDH activity was increased, while the activities of other respiratory chain-linked enzymes, SDH, MQO and D-LDH, were not changed (Table 1). Table. 1. Several dehydrogenase activities in the membrane of KY9714, ndh disruptant and ndh over-expressed strains grown on glucose minimum medium. Dehydrogenases Membrane-bound NADH Succinate Malate L-Lactate D-Lactate Cytoplasmic Malate Lactate Isocitrate Specific activity (U/mg) KY9714 ndh disruptant ndh over-expressed 8.53 ± 2.64 (4) 0.73 ± 0.11 (5) 0.45 ± 0.09 (2) 0.14 ± 0.04 (4) 0.17 ± 0.02 (4) 1.98 ± 0.43 (5) 0.72 ± 0.27 (3) 0.52 ± 0.08 (5) 0.03 ± (4) 0.72 ± 0.02 (5) 0.38 ± 0.08 (2) 0.30 ± 0.05 (4) 0.16 ± 0.04 (4) 1.37 ± 0.20 (5) 0.91 ± 0.33 (3) 0.38 ± 0.04 (5) 77.0 ± 15.6 (4) 0.61 ± 0.07 (5) 0.10 ± 0.03 (4) 0.04 ± 0.01 (4) 0.08 ± 0.01 (4) 1.27 ± 0.44 (4) 0.53 ± 0.13 (2) 0.39 ± 0.07 (5) Whereas, L-LDH and MQO activities were largely decreased in the over-expressed strain, but SDH and D-LDH were not much altered. In addition, a reasonably high LDH activity present in the cytoplasmic fraction was a little increased and decreased in the disruptant and the overexpressed strains, respectively (Table 1). These results suggest that L-LDH and LDH coupling may operate as a NADH re-oxidation system in addition to MQO and MDH coupling system as speculated by Molenaar et al. (2000) in C. glutamicum (Fig 1). Such coupling reactions were reproduced by reconstitution of the cytoplasmic and membrane fractions prepared from the ndh disruptant. Although the NADH oxidation activity was higher with the MQO-MDH system than with the L-LDH-LDH system, the large activity change of the L-LDH-LDH system in both the disruptant and over-expressed strains suggests that the L-LDH-LDH system seems to contribute very much for the NADH re-oxidation. The NADH re-oxidation systems may also affect the operation of TCA cycle. A high operation of such coupling systems for NADH oxidation might lead to a lower accumulation of oxaloacetate or pyruvate, which could cause a lower production of an important precursor for the anabolism of many amino acids, including glutamate. The opposite situation could be observed in the over-expressed strain, in which the coupling system

5 may not be functioning. Thus, manipulation of NDH-2 might be useful to control the central metabolism. NAD(P)H NDH-2 2H + /O L-lactate SDH bd O 2 4H + /O 2H + /O NADH LDH pyruvate L-LDH MQ bc 1 c aa 3 O 2 NAD MDH malate OAA D-LDH MQO cyanide-resistant bypass oxidase O 2 Fig. 1. Schematic representation of hypothetical respiratory chain of C. glutamicum (Nantapong et al., 2004) 2. Characterization of C. glutamicum NADH dehydrogenase NDH-2 was first purified from a lysozyme-sensitive strain of C. glutamicum and shown to exhibit NADPH oxidase activity in addition to NADH dehydrogenase activity (Matsushita et al., 2001). Further characterization of this enzyme has been performed by purifying it from NDH-2 over-producing strain (Nantapong et al., 2005). Similar to NDH-2 found in other organisms, the spectrum of the purified enzyme showed that C. glutamicum NDH-2 contains flavin moiety. This is consistent with the sequence information of this enzyme, which shows that it contains an FAD binding motif. In addition, we also presented the evidence that the flavin moiety of C. glutamicum NDH-2 is a non-covalently bound FAD, but not covalently bound FMN. Although E. coli NDH-2 contains thiolate-bound Cu(I) and is predicted to have two conserved cysteine residues, C. glutamicum NDH-2 does not contain Cu and also has neither heavy-metal-associated domain nor the conserved cysteine residues in its polypeptide sequence. NDH-2 of E. coli and external alternative NADH dehydrogenase of N. crassa contain the predicted transmembrane domain at the C-terminal and the N-terminal region, respectively, while NDH-2 of Acidianus ambivalens contains three possible putative amphipatic helices, which are also present in E. coli NDH-2. Therefore, the secondary structure of C. glutamicum NDH-2 was examined, which revealed that there is a single putative transmembrane region in the C-terminal region (around amino acid residues 384~439), and also similar possible amphipatic regions (residues 179~196, 329~346 and 442~459). When the oxidation of NAD(P)H was examined with various electron acceptors, the reduction of MD, Q 1, Q 2 and FR was observed with both NADH and NADPH whereas the

6 reduction of MQ 2 was only observed with NADH (Fig. 2). The K m and V max for MQ 2 reduction of C. glutamicum NDH-2 with NADH (K m = 5 µm; V max = 82 U/mg) were reasonably low and high, respectively, whereas the affinity (K m = 41 µm) for ubiquinone analogue, Q 1, is significantly lower than that in E. coli NDH-2 (K m = 5.9 µm). And also the K m values for Q 1, Q 2 and FR reduction due to NADH oxidation were higher than those due to NADPH oxidation, and thus these artificial electron acceptors are more favorable electron acceptors for coupling with NADPH oxidation. The results suggest that menaquinone is a more favorable electron acceptor for C. glutamicum NDH-2 than other quinones at least for NADH oxidation. Furthermore, since the purified enzyme exhibits reasonably higher oxidase activity when coupled with NADPH, the physiological electron acceptor coupled with NADPH oxidation of C. glutamicum NDH-2 would be oxygen, although the actual affinity for oxygen was not determined in this study. Taken together with these results, it can be concluded that NDH-2 in C. glutamicum oxidizes NADH tightly coupled with a natural electron acceptor, menaquinone, within the membrane, while it may oxidize NADPH partly coupled with oxygen reduction. However, the results obtained with lanzoparzole and antimycin A inhibition provided the evidence that C. glutamicum NDH-2 donates electrons from both NADH and NADPH to quinone at the same manner, or at the same site, yet different efficiency and that oxygen is the most preferable electron acceptor for NADPH oxidation than quinones. In conclusion, the binding of NADPH and/or NADH may cause a conformational change in the enzyme structure, which causes a change in the binding site for the electron acceptor, including oxygen, or may directly overlap the acceptor binding site so that NADPH binding instead of NADH changes the binding of acceptors. U/mg NAD(P)H oxidase ph U/mg NAD(P)H:Q ph U/mg 35 NAD(P)H:MQ 2 :FR ph Fig. 2 ph-dependent NAD(P)H oxido-reductase activities with various artificial electron acceptors (Q2, or MQ) or oxygen. The closed and opened circles represent the oxidation of NADH and NADPH, respectively.

7 Various redox carriers of the electron transport chain are a possible source of reactive oxygen species formation. In mammalian mitochondria, the main sites for superoxide and hydrogen peroxide production are NADH:quinone reductase (Complex I) and ubiquinol: cytochrome c reductase. NDH-2 of E. coli, T. brucei or S. cerevisiae is also a source of the endogenous superoxide production. It has been shown that NDH-2 of C. glutamicum is a potential source of superoxide and hydrogen peroxide formation (Fig. 3) (Nantapong et al., 2005). The oxidation of NADH and NADPH with oxygen, especially NADPH oxidation, with NDH-2 led to the formation of superoxide and hydrogen peroxide. Since NDH-2 of C. glutamicum has a high ability to oxidize NADPH, it is reasonable to generate such a Intensity A high level of superoxide from NADPH. Whereas, E. coli enzyme can not oxidize NADPH so well that it generates superoxide not as high an amount as observed for NDH-2 from C. glutamicum. In contrast, a similar small amount of superoxide formation from NADH was observed by EPR between both NDH-2 from C. B glutamicum and E. coli, in our laboratory. However, similar to C. glutamicum enzyme, NDH-2 of T. brucei and S. cerevisiae produce a relatively high superoxide from NAD(P)H and NADH, respectively (Fang and C Beattie, 2002; 2003). Fig. 3. EPR determination of its superoxide radical produced by purified NDH-2. Approximately 1.2 µm purified NDH-2 was incubated for 15 min with 1 mm NADPH (A and B) or NADH (C and D), 0.2 µm FAD and 45 mm DEPMPO in 50 mm Na-acetate buffer, ph 5.5, (A and C) or in 50 mm KPB, ph 6.5, (B and D). D [G] NDH-2 of C. glutamicum exhibits a unique character, NADPH oxidation, which occurs more often in eukaryotic enzymes than in prokaryotic ones. The external alternative NADH dehydrogenases NDE-1 and NDE-2 of N. crassa oxidize NADPH at acidic ph similar to the C. glutamicum enzyme (Carneiro et al., 2004; Melo et al., 2001). Therefore, a phylogenetic tree of

8 NDH-2 from various organisms including C. glutamicum was constructed using Clustal W program. As expected, NDH-2 of Corynebacterium sp. as well as the counterpart Mycobacterium sp. were found to be more closely related to eukaryotic enzymes than other prokaryotic ones. Thus, NDH-2 of C. glutamicum seems to be evolved from the same ancestor as the enzymes found in yeast and fungi. NDH-2 of C. glutamicum donates electrons to membranous menaquinone at least when it reacts with NADH, while it may more preferentially transfer electrons to oxygen with NADPH concomitant with the production of superoxide or hydrogen peroxide. C. glutamicum cells contain a relatively high amount of superoxide dismutase as well as catalase, and the superoxide dismutase seems to be localized both in the cytoplasm and periplasm. Thus, NDH-2 of C. glutamicum may work as an NADH dehydrogenase as a primary dehydrogenase of the NADH oxidase respiratory chain and also as an NADPH oxidase not coupled with the respiratory chain. References Bott, M., Niebisch, A. (2003) The respiratory chain of Corynebacterium glutamicum. J. Biotech. 104: Carneiro, P., Duarte, M., Videira, A. (2004) The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria. Biochim. Biophys. Acta 1608: Fang, J., Beattie, D.S. (2002) Rotenone-insensitive NADH dehydrogenase is a potential source of superoxide in procyclic Trypanosoma brucei mitochondria. Mol. Biochem. Parasitol. 123: Fang J., Beattie, D.S. (2003) External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide. Free Radical Biol. Med. 34: Matsushita, K., Ohnishi, T., Kaback, R. (1987) NADH-ubiquinone oxidoreductase of the Escherichia coli aerobic respiratory chain. Biochemistry 26: Matsushita, K., Otofuji, A., Iwahashi, M., Toyama, H., Adachi, O. (2001) NADH dehydrogenase of Corynebacterium glutamicum. Purification of NADH dehydrogenase II homologue able to oxidize NADPH. FEMS Microbiol. Lett. 204: Melo, A.M., Duarte, M., Moller, I.M., Prokisch, H., Dolan, P.L., Pinto, L., Nelson, M.A., Videira, A. (2001) The external calcium-dependent NADPH dehydrogenase from Neurospora crassa mitochondria. J. Biol. Chem. 276: Molenaar, D., van der Rest, M.E., Drysch, A., Yucel, R. (2000) Functions of the membrane-associated and cytoplasmic malate dehydogenases in the citric acid cycle of Corynebacterium glutamicum. J. Bacteriol. 182: Nantapong, N., Kugimiya, Y., Toyama, H., Adachi, O., Matsushita, K. (2004) Effect of NADH dehydrogenase-disruption and over-expression on the respiratory-related metabolism in Corynebacterium glutamicum KY9714. Appl. Microbiol. Biotechnol. 66: Nantapong N., Otofuji A., Migita CT., Adachi O., Toyama H., Matsushita K. (2005) Electron transfer ability from NADH to menaquinone and from NADPH to oxygen of type II NADH dehydrogenase of Corynebacterium glutamicum. Biosci. Biotechnol. Biochem. 69: