Cytochromes P450 catalyze oxidation of α,β-unsaturated aldehydes

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Abstract

We sought to establish whether heme-thiolate monooxygenases oxidize, α,β-unsaturated aldehydes generated during lipid peroxidation. Several recombinant P450s co-expressed with NADPH:P450 oxidoreductase were surveyed for aldehyde oxidation activity with anthracene-9-carboxaldehyde and 4-hydroxy-trans-2-nonenal (HNE). Murine P4502c29, human P4503A4, human P4502B6, and rabbit P4502B4 were good catalysts of aldehyde oxidation to carboxylic acids. Other P450s (e.g., P4501A2, 2E1, and 2J2) did not oxidize these aldehydes. P4502c29 and P4503A4 displayed Km/S0.5 values of approx. 1–20 μM. The product measured by HPLC that co-migrates with authentic 4-hydroxynonenoic acid (HNA) had a mass spectrum identical to the standard. Using P4502c29, HNE was a mixed-competitive inhibitor of anthracene-9-carboxaldehyde oxidation, suggesting that both aldehydes are substrates for P4502c29. Specific inhibitors of aldehyde dehydrogenases and P450 were used to assess their role in the metabolism of HNE in primary rat hepatocytes. Inhibitors of aldehyde dehydrogenase (cyanamide) inhibited HNA formation by 60% and together cyanamide and miconazole (P450) caused over 85% inhibition of HNA formation. P450s are significant participants in metabolism of endogenous and exogenous unsaturated aldehydes in primary rat hepatocytes.

Section snippets

Chemicals and plasmids

An expression plasmid for murine P4502c29 was provided by Goldstein, National Institutes of Environmental Health Sciences, Research Triangle Park, NC [17]. The NADPH:P450 oxidoreductase expression plasmid was provided by Mark Doll and David Hein, Department of Pharmacology and Toxicology, University of Louisville School of Medicine. Preparations of Escherichia coli membranes containing recombinant NADPH:P450 oxidoreductase and either P4501A2, 2B6, 2E1, or 3A4 were prepared as described [18]. In

Evaluation of P450s capable of catalyzing anthracene-9-carboxaldehyde oxidative metabolism to its carboxylic acid product

Watanabe et al. [31] showed that P4502c29 is an aldehyde oxygenase utilizing a cannabinoid aldehyde at low μM concentrations as a substrate. Therefore, we sought to evaluate the ability of a number of P450s for their ability to oxidize a synthetic aldehyde substrate, 9-AA [27], [28]. Several P450s rapidly metabolized the aldehyde compound, while others apparently did not oxidize this aldehyde to its carboxylic acid (Table 1). For example, 9-AA oxidation activity for human P4503A4, human 2B6,

Discussion

A typical reaction catalyzed by P450s is the hydroxylation of an unfunctionalized alkyl group. However, examples are also known in which P450s catalyze the oxidation of aldehydes to acids with the incorporation of molecular oxygen into the products [36]. Several critical P450-catalyzed reactions have been shown to involve oxidation of aldehydes or hemiacetals, namely the aromatase and sterol 14α-demethylase reactions. The formation of estrogen requires oxidation of the 19-methyl group of

Acknowledgments

Supported by USPHS Grant P01 ES11860 (RAP/AB). The authors express appreciation for K. Cameron Falkner for his helpful discussions about biochemical kinetic analysis.

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