Cytochrome P450: taming a wild type enzyme

Abstract

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable' - they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability.

Figure 1

P450 BM3 has been engineered to hydroxylate a broad spectrum of molecules of different molecular weights. Molecular weights range from 30 for ethane (1) to 330 for 11-α-hydroxyprogesterone (4). Propane (2) and ibuprofen methyl ester (3) are also readily hydroxylated. Arrows denote sites of hydroxylation.

Figure 2

Crystal structure showing the residues of P450 BM3 altering substrate recognition and thermostability. (a) Stick representations are shown of 15 active site residues distal to the heme which can undergo mutation and alter substrate scope and selectivity. (b) Residues that improve thermostability are colored red on the crystal structure (PDB: 1BU7).