Flavocytochrome P450 BM3: An update on structure and mechanism of a biotechnologically important enzyme

Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner – a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved. Introduction Since their discovery in the 1950s, the P450s (cytochromes P450) have been studied in enormous detail due to their involvement in a plethora of crucial cellular roles – from human drug and steroid metabolism through to the bacterial catabolism of unusual compounds as energy sources [1,2]. The P450s are haem b-containing mono-oxygenase enzymes, often catalysing hydroxylation of hydrophobic substrate molecules. Molecular oxygen is bound to the haem iron and reductively activated to produce an oxyferryl radical cation intermediate. This ‘active oxygen’ compound I species attacks a substrate bound adjacent to the haem. Recent studies suggest that a preceding intermediate in the catalytic cycle (the ferric peroxy form or compound 0) may also be catalytically competent, at least with respect to epoxidation across carbon– carbon double bonds [3]. However, computational studies indicate that compound I is a superior oxidant even for alkene epoxidation [4]. The key to the P450s’ ability to cleave molecular oxygen is the axial ligation of haem iron by a conserved cysteine residue in the protein. The thiolate ligation is shared among the P450, NOS (nitric oxide synthase) and chloroperoxidase enzyme families – members of which all activate oxygen [5]. The structure and cellular roles of P450