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Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria

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Abstract

Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O2. When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O2-free N2 carrier-gas) to microoxic (sparged with 3% O2/97% N2 mixed carrier-gas) growth conditions in the mid exponential phase (OD660=1.0). When the strain grew under 3% O2/97% N2, the medium remains anoxic. Thirty minutes after beginning aeration with 3% O2, the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H2O, and the estimated K m for oxygen was 61.9 μM. These data demonstrate that an O2-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.

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Abbreviations

NRIC:

NODAI Research Institute-Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

PMSF:

phenylmethylsulfonyl fluoride

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Acknowledgments

We thank Dr Takeshi Nishino, Dr Kenji Ohnishi, and Dr Tomoyuki Nakagawa for helpful suggestion and valuable discussion, and Mr Yusuke Watamura and Mr Satoshi Nakayama, Mr Masaki Ono, Mr Tetsuya Matsumoto, and Mr Tatsuya Noguchi for helpful technical assistance at Tokyo University of Agriculture.

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Authors and Affiliations

  1. Department of Biosciences, Tokyo University of Agriculture, 1-1-1 Setagaya-ku, Tokyo, 156-8502, Japan

    Shinji Kawasaki, Jun Ishikura, Daisuke Chiba & Youichi Niimura

  2. Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan

    Tomoko Nishino

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  1. Shinji Kawasaki
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  2. Jun Ishikura
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  3. Daisuke Chiba
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  4. Tomoko Nishino
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  5. Youichi Niimura
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Correspondence to Shinji Kawasaki.

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Kawasaki, S., Ishikura, J., Chiba, D. et al. Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria. Arch Microbiol 181, 324–330 (2004). https://doi.org/10.1007/s00203-004-0659-3

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  • DOI: https://doi.org/10.1007/s00203-004-0659-3

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