Purified thioredoxin reductase from O2-sensitive Bifidobacterium bifidum degrades H2O2 by interacting with alkyl hydroperoxide reductase.

Bifidobacterium is beneficial for host health and exhibits different O2 sensitivity levels among species or strains via unknown mechanisms. Bifidobacterium bifidum JCM1255T, a type species of Bifidobacterium, is an O2-sensitive bacterium that can grow under low-O2 (5%) conditions, and the growth of this species is inhibited under high-O2 conditions (10% ∼) with accumulation of H2O2. We previously reported that NADH or NAD(P)H oxidase-active fractions were detected during purification using microaerobically grown B. bifidum cells, and the active enzyme was purified from the NADH oxidase-active fraction. The purified enzyme was identified as b-type dihydroorotate dehydrogenase (DHODb) and characterized as a dominant H2O2 producer in B. bifidum. In this study, we performed further purification of the enzyme from the NAD(P)H oxidase-active fraction and characterized the purified enzyme as a part of the H2O2 degradation system in B. bifidum. This purified enzyme was identified as thioredoxin reductase (TrxR); the NAD(P)H oxidase activity of this enzyme was not expressed in anaerobically grown B. bifidum, and mRNA expression was induced by O2 exposure. Furthermore, the purified B. bifidum TrxR interacted with recombinant alkyl hydroperoxide reductase (rAhpC) and exhibited NAD(P)H peroxidase activity. These results suggest that TrxR responds to O2 and protects B. bifidum from oxidative stress by degrading H2O2 via the TrxR-AhpC system.

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.