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Journal of Bacteriology, September 2000, p. 5046-5051, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in Amphibacillus xylanus

Youichi Niimura,1,* Yoshitaka Nishiyama,1 Daisuke Saito,1 Hirokazu Tsuji,1 Makoto Hidaka,2 Tatsurou Miyaji,3 Toshiro Watanabe,3 and Vincent Massey4

Department of Bio-Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-85027,1 Department of Biotechnology, The University of Tokyo, Tokyo 113,2 and Department of Food Science and Technology, Tokyo University of Agriculture, Abashiri-shi, Hokkaido 099-2493,3 Japan, and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-06064

Received 6 March 2000/Accepted 6 June 2000

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta -NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 × 106 M-1 s-1. Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH2 reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH2 is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The Vmax values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s-1. The Km values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.

* Corresponding author. Mailing address: The Department of Bio-Science, Tokyo University of Agriculture, 1-1-1 Setagaya-ku, Tokyo 156-85027, Japan. Phone: 03-5477-2761. Fax: 03-5477-2668. E-mail: niimura{at}nodai.ac.jp.

Journal of Bacteriology, September 2000, p. 5046-5051, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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