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Applied and Environmental Microbiology, February 2009, p. 629-636, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.02111-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

b-Type Dihydroorotate Dehydrogenase Is Purified as a H2O2-Forming NADH Oxidase from Bifidobacterium bifidum{triangledown} ,{dagger}

Shinji Kawasaki,§* Takumi Satoh,§ Mitsunori Todoroki, and Youichi Niimura

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

Received 11 September 2008/ Accepted 28 November 2008

Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O2-sensitive and microaerophilic species is due to the existence of a system to produce H2O2 in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H2O2. Bifidobacterium bifidum, an O2-sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H2O2-forming NADH oxidase reaction in the presence of O2. The kinetic parameters suggested that the enzyme could be involved in H2O2 production in highly aerated environments.

* Corresponding author. Mailing address: Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Setagaya-ku, Tokyo 156-8502, Japan. Phone and fax: 81-3-5477-2764. E-mail: kawashin{at}nodai.ac.jp

{triangledown} Published ahead of print on 5 December 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

§ Both authors contributed equally to this work and are co-first authors.

Applied and Environmental Microbiology, February 2009, p. 629-636, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.02111-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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