A FLAVOPROTEIN FUNCTIONAL AS NADH OXIDASE FROM AMPHIBACILLUS-XYLANUS EP01 - PURIFICATION AND CHARACTERIZATION OF THE ENZYME AND STRUCTURAL-ANALYSIS OF ITS GENE

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolate d, shows rapid aerobic growth even though it lacks a respiratory pathw ay. Thus, the oxidative consumption of NADH, produced during glycolysi s and pyruvate oxidation, should be especially important for maintenan ce of intracellular redox balance in this bacterium. We purified a fla voprotein functional as NADH oxidase from aerobically growing A.xylanu s Ep01. The A.xylanus enzyme is a homotetramer composed of a subunit ( M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enz yme catalyzes the reduction of oxygen to hydrogen peroxide with beta-N ADH as the preferred electron donor and exhibits no activity with NADP H. The flavoprotein gene of A. xylanus Ep01 was cloned by using a spec ific antibody. The amino acid sequence of 509 residues, deduced from t he nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhim urium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN- 1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no r espiratory chain, grow equally well under both aerobic and anaerobic c onditions, which distinguishes these two genera. Salmonella spp., whic h are gram-negative bacteria, are taxonomically distant from gram-posi tive bacteria such as Bacillus spp. and Amphibacillus spp. The above f indings, however, suggest that the flavoprotein functional as NADH oxi dase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase di verged recently, with only small changes leading to their functional d ifferences.