Molecular Cloning and Sequence Analysis of the Gene Encoding the H<sub>2</sub>O-forming NADH Oxidase from<i>Streptococcus mutans</i>

Matsumoto, Junichi; Higuchi, Masako; Shimada, Muneaki; Yamamoto, Yoshikazu; Kamio, Yoshiyuki

doi:10.1271/bbb.60.39

Cited by 44 publications

(30 citation statements)

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“…The consensus sequences were clearly discernible: the FADbinding site motif GXT(H/S)AG in position 8 ± 14 (counted from the bnox N-terminus), the putative catalytic cysteine residue in position 42, and the NADbinding site GXGYIG in positions 156 ± 161. Alignment with the sequences of the NADH oxidases of Enterococcus faecalis [30] and Streptococcus mutans [32] demonstrate at most 34% identity between any two including the two novel proteins, except for 55% between sfnox and the enzyme from E. faecalis.…”

Section: Sequence Analysismentioning

confidence: 99%

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

et al. 2002

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Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD+ can be considered solved, the inverse problem of regenerating NAD+ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD+ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O2 either to undesirable H2O2 or to innocuous H2O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O2 to H2O.

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Section: Sequence Analysismentioning

confidence: 99%

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

et al. 2002

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“…Two types of NADH oxidases have indeed been described, the first one catalyzing the four-electron reduction of O 2 into 2 H 2 O (13,17,19,21) and the second the two-electron reduction of O 2 to H 2 O 2 (2,10,11,24). These enzymes are present in several lactic acid bacteria that possess either a NADH-H 2 O 2 or a NADH-H 2 O oxidase and sometimes both (5, 7).…”

Section: Fig 1 Effect Of Aeration On the Growth Of L Delbrueckii Smentioning

confidence: 99%

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

Marty-Teysset

Torre

Garel

2000

Appl Environ Microbiol

121

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The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H 2 O 2 oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) is an important species of lactic acid bacteria currently used in the industrial production of fermented milk products. L. delbrueckii subsp. bulgaricus is an aerotolerant anaerobe that obtains most of its energy from homolactic fermentation (12). It does not require strict anaerobic growth conditions and tolerates the concentration of O 2 in air. Even though L. delbrueckii subsp. bulgaricus does not use O 2 in its energetic metabolism, it is likely that the presence of oxygen in its environment can influence its physiology. Indeed, some lactic acid bacteria possess oxidases that utilize molecular oxygen to oxidize substrates such as pyruvate (22) or NADH (2,5,8,16,(23)(24)(25). As these oxidation reactions cannot occur under anaerobic conditions, metabolism in the presence of oxygen cannot be identical to that in the absence of oxygen. Also, the activities of these oxidases can produce partially reduced oxygen species such as the superoxide radical (O 2 .Ϫ ), hydrogen peroxide (H 2 O 2 ), the hydroxyl radical (HO . ), and other peroxyl radicals or peroxides that will cause an oxidative stress in the cell. It is therefore expected that the presence of oxygen will induce a specific cellular response to such oxidative stress. In this work, a difference in the growth of L. delbrueckii subsp. bulgaricus in the presence and absence of oxygen was observed. It was also found that L. delbrueckii subsp. bulgaricus could reduce oxygen into hydrogen peroxide with an NADH oxidase, probably to eliminate the oxygen present. However, this detoxification of oxygen led to an overproducti...

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“…The observed homoacetic fermentation at low D under aerobic conditions has been explained as a result of four mechanisms operating simultaneously, all driving pyruvate conversion via PDH. However, at higher dilution rates (D = 0.3 h 31 ), PDH could accommodate a much higher pyruvate £ux than would expected from previously reported data obtained with L. lactis MG1363 [10] ( Table 2, results in parentheses). This higher acetate production results from the relief of the reported NADH inhibition on PDH activity [11] as a consequence of an e¤cient NOX overproduction (Table 2).…”

Section: Discussionmentioning

confidence: 50%

“…lactis strain NZ9800 [7], harboring plasmid pNZ2600, was used throughout this work. Plasmid pNZ2600 is a derivative of pNZ8020 containing the water-forming NOX-encoding gene of Streptococcus mutans, nox-2 [10], under control of the lactococcal nisA promoter [3,4]. In the appropriate lactococcal background (strain NZ9800) this recombinant plasmid allowed the nisin-controlled expression of the encoded NOX [9].…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

Felipe

1999

FEMS Microbiology Letters

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The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADHoxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via K-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains. ß

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Molecular Cloning and Sequence Analysis of the Gene Encoding the H₂O-forming NADH Oxidase fromStreptococcus mutans

Cited by 44 publications

References 0 publications

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

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Molecular Cloning and Sequence Analysis of the Gene Encoding the H2O-forming NADH Oxidase fromStreptococcus mutans

Cited by 44 publications

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Cofactor Regeneration of NAD+from NADH: Novel Water‐Forming NADH Oxidases

Cofactor Regeneration of NAD+from NADH: Novel Water‐Forming NADH Oxidases

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

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Molecular Cloning and Sequence Analysis of the Gene Encoding the H₂O-forming NADH Oxidase fromStreptococcus mutans

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases