Dithionite-supported hydroxylation of palmitic acid by cytochrome P450BM-3.
The ability of dithionite, an inexpensive reducing agent routinely used to produce the ferrous-carbonyl form of P450, to support P450BM-3-catalyzed hydroxylation of palmitate was studied. The hydroxylation products in the presence of dithionite were 15, 14, and 13-hydroxyhexadecanoate, with relative distributions similar to those observed with NADPH. The hydroxylation reaction was carried out in two separate steps, anaerobic reduction and subsequent oxidation of P450BM-3 by oxygen bubbling. The reduction step was much slower than the oxidation step, thus limiting the overall rate of hydroxylation. Upon addition of dithionite, the reductase domain of P450BM-3 seemed to be reduced before significant reduction of the heme domain occurred. The discovery of new reducing agents for P450-catalyzed reaction raises the possibility of replacing NADPH in specialty chemical hydroxylation catalyzed by P450s, especially catalytically self-sufficient P450s, such as P450BM-3 or recombinant fusion proteins of P450 covalently linked to a reductase.
Fang
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Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
Halpert
J R
JR
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004227:Dithionite / Q000494:pharmacology*; D006900:Hydroxylation; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D019308:Palmitic Acid / Q000378:metabolism*
D001426:Bacterial Proteins; D004227:Dithionite; D019308:Palmitic Acid; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases