Protein engineering of cytochrome P450cam

The cytochrome P450 superfamily of heme monooxygenases catalyse many reactions involved in the biosynthesis and degradation of endogenous compounds and in the oxidative metabolism of xenobiotics. These enzymes all share the same overall catalytic mechanism, and their varied substrate specificities arise from differences in their active site topologies, suggesting that it should be possible to engineer P450 enzymes for the oxidation of unnatural substrates. A review of the active site protein engineering experiments on cytochrome P450cam reported to date is presented here. Cytochrome P450cam is a stable, soluble enzyme isolated from Pseudomonas Putida, and is an excellent system for rational protein engineering. The current understanding of the details of the P450cam catalytic mechanism, including the uncoupling pathways, is first discussed. The factors controlling substrate access, binding and turnover activity, and the coupling efficiency have been investigated by site-directed mutagenesis. The results of this work have enabled the rational redesign of P450cam to broaden the substrate range, to improve the activity and coupling efficiency towards unnatural substrates, and to engineer the regioselectivity of substrate oxidation. The construction of a “self-sufficient” fusion protein containing all three proteins of the P450cam system brings one step nearer the in vitro and in vivo biotechnological applications of P450cam.

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