Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.
Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone.
Weber
F J
FJ
Department of Food Science, Agricultural University, Wageningen, The Netherlands.
van Berkel
W J
WJ
Hartmans
S
S
de Bont
J A
JA
D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids; D004578:Electron Spin Resonance Spectroscopy; D004852:Epoxy Compounds / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000187:drug effects; D011136:Polysorbates / Q000494:pharmacology; D013455:Sulfur / Q000032:analysis
D000475:Alkenes; D000596:Amino Acids; D004852:Epoxy Compounds; D007506:Iron-Sulfur Proteins; D011136:Polysorbates; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D013455:Sulfur; D007501:Iron; D010105:Oxygenases; D009247:NADH, NADPH Oxidoreductases