Purification and analysis of a flavoprotein functional as NADH oxidase from Amphibacillus xylanus overexpressed in Escherichia coli.
The gene encoding the Amphibacillus xylanus flavoprotein has been cloned into pTTQ18 and overexpressed in Escherichia coli. The recombinant enzyme has been purified to homogeneity yielding 15 mg of pure enzyme/liter of cell culture. Recombinant flavoprotein is fully active and has an absorption spectrum identical to that of the enzyme purified from A. xylanus. The N-terminal sequence analysis and analytical gel filtration data confirm the structural identity of recombinant and A. xylanus enzymes. The Km value for oxygen and the Km value for NADH are 1.7 mM and 33.3 microM, respectively. In the presence of free additional FAD, however, the Km value for oxygen decrease dramatically. The NADH oxidase activity is accelerated markedly in the presence of additional FAD. The intracellular free FAD concentration of A. xylanus is calculated about 13 microM. This FAD concentration would be enough to accelerate the NADH oxidase activity of flavoprotein in cells of A. xylanus. Two-electron reduction of the enzyme FAD by the strong reductant dithionite occurs during the total uptake of 6 electrons. Such behavior usually indicates the presence of non-flavin redox centers. The high degree of homology between this enzyme and alkyl hydroperoxide reductase F52a protein and thioredoxin reductase suggests that these centers are the redox-active disulfide adjacent to the FAD and another disulfide, which is able to slowly interchange with the redox-active disulfide. The presence of two disulfides has been demonstrated.
Ohnishi
K
K
Department of Agricultural Chemistry, Tokyo University of Agriculture, Japan.
Niimura
Y
Y
Yokoyama
K
K
Hidaka
M
M
Masaki
H
H
Uchimura
T
T
Suzuki
H
H
Uozumi
T
T
Kozaki
M
M
Komagata
K
K
Nishino
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D002384:Catalysis; D003545:Cysteine / Q000032:analysis; D004227:Dithionite / Q000737:chemistry; D005415:Flavins / Q000378:metabolism; D005420:Flavoproteins / Q000302:isolation & purification*; D007700:Kinetics; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry
D005415:Flavins; D005420:Flavoproteins; D008670:Metals; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D009243:NAD; D004227:Dithionite; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine