Abstract
Cytochrome P450 (P450) 2S1 is one of the "orphan" P450s. Although it has been clearly demonstrated to catalyze reductive reactions, its role in NADPH-dependent oxidations has been ambiguous. We utilized an untargeted LC-MS metabolomic approach with recombinant human P450 2S1 and extracts of rat stomach and intestine, sites of P450 2S1 localization. The search yielded several candidates, including the product 19-hydroxyarachidonic acid. Subsequent 18O analysis and in vitro studies with commercial arachidonic acid and 19-hydroxyarachidonic acid were used to validate ω-1 hydroxylation as an NADPH- and O2-dependent reaction. Steady-state kinetic assays were done for ω-1 hydroxylation reactions of P450 2S1 with arachidonic, linoleic, ω-linolenic, eicosapentaenoic, and docosapentaenoic acids. The rates of hydroxylation were slow, but no detectable activity was seen with either medium-chain length or saturated fatty acids. We conclude that P450 2S1 is a fatty acid ω-1 hydroxylase, although the physiological relevance of these oxidations remains to be established. The metabolomic approach is feasible for orphan P450s and other enzymes, in regard to annotation of function.
SIGNIFICANCE STATEMENT An untargeted mass spectrometry approach was utilized to identify ω-1 hydroxylation of arachidonic acid as an oxidative reaction catalyzed by human cytochrome P450 2S1. The enzyme also catalyzes the relatively slow ω-1 hydroxylation of several other unsaturated long-chain fatty acids.
- arachidonic acid/eicosanoids
- cytochrome P450
- enzyme kinetics
- fatty acids
- HPLC
- mass spectrometry/MS
- metabolomics
- NADPH cytochrome P450 reductase
- UPLC
- The American Society for Pharmacology and Experimental Therapeutics