Further characterization of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.
Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed alpha-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 microM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2 mM, but CO did not. Imidazole, an inhibitor of plant alpha-oxidation, showed no inhibitory effect at 1 mM.
Matsunaga
I
I
Section of Molecular Regulation, Toneyama Institute for Tuberculosis Research, Osaka City University Medical School, Toyonaka, Osaka, 560-0045, Japan. matsunagai@msic.med.osaka-cu.ac.jp
Yamada
M
M
Kusunose
E
E
Miki
T
T
Ichihara
K
K
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C019081:fatty acid alpha-hydroxylase