Measurement of P450 difference spectra using intact cells

Abstract

Whole-cell assays provide a rapid means of determining expression and substrate binding for cytochrome P450 enzymes expressed heterologously in Escherichia coli and, potentially, other host cells. Such assays are particularly useful for screening large libraries of mutant P450s, where rapid, high-throughput assays are needed for first-tier screens that can, firstly, quantify any P450 form independent of P450 subfamily and, secondly, suggest possible ligands before more labor-intensive direct measurement of substrate turnover. Whole-cell spectral techniques are derived from methods that have been used for a long time to study P450s in microsomal or other subcellular fractions (Omura T and Sato R, J Biol Chem 239:2370-2378, 1964; Schenkman JB et al., Biochemistry 11:4243-4251, 1972), but recent studies have detailed important modi fications which allow quantitative results to be obtained in whole cells (Otey CR, Methods in Molecular Biology, vol. 230, Humana, Totowa, NJ, pp. 137-139, 2003; Johnston WA et al., J Biomol Screen 13:135-141, 2008). A general method is presented here for the measurement of difference spectra on recombinant P450 cultures that can be applied to both carbon monoxide and any number of alternative ligands that alter the characteristic spectral signature of P450s. © Springer Science+Business Media New York 2013.