b-type dihydroorotate dehydrogenase is purified as a H2O2-forming NADH oxidase from Bifidobacterium bifidum.
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 O(2)-sensitive and microaerophilic species is due to the existence of a system to produce H(2)O(2) 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 H(2)O(2). Bifidobacterium bifidum, an O(2)-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 H(2)O(2)-forming NADH oxidase reaction in the presence of O(2). The kinetic parameters suggested that the enzyme could be involved in H(2)O(2) production in highly aerated environments.
Kawasaki
Shinji
S
Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan. kawashin@nodai.ac.jp
Satoh
Takumi
T
Todoroki
Mitsunori
M
Niimura
Youichi
Y
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002853:Chromatography, Liquid; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D000090722:Dihydroorotate Dehydrogenase; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009876:Operon; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D021122:Protein Subunits; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D000090722:Dihydroorotate Dehydrogenase; D009097:Multienzyme Complexes; D021122:Protein Subunits; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases