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First published on March 21, 2006; DOI: 10.1124/mol.106.023648

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69/6/2007    most recent
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Received for publication February 17, 2006.
Revised March 21, 2006.
Accepted for publication March 21, 2006.

Recombinant enzymes over-expressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1

Zhong-Liu Wu 1, Christal D. Sohl 1, Tsutomu Shimada 1, F. Peter Guengerich 1*

1 Vanderbilt University School of Medicine

* Address correspondence to: E-mail: f.guengerich{at}vanderbilt.edu

Abstract

Human cytochrome P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochrome P450s have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochrome P450s from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol cytochrome P450/liter culture, the highest level achieved in this laboratory to date. Assays with a number of the typical P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (kcat 3.8 min-1) and also arachidonic acid oxidation, albeit at a very low rate (~ 0.05 min-1). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several pro-carcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of pro-carcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of pro-carcinogens could lead to accumulation of mutations and enhance the carcinogenic process.


Key words: Cytochrome P450, Enzymology, Structure/function/mechanism


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