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 isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium 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 respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.
Niimura
Y
Y
Department of Food Science and Technology, Tokyo University of Agriculture, Japan.
Ohnishi
K
K
Yarita
Y
Y
Hidaka
M
M
Masaki
H
H
Uchimura
T
T
Suzuki
H
H
Kozaki
M
M
Uozumi
T
T
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010101:Oxygen Consumption; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014461:Ultracentrifugation
D005420:Flavoproteins; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases