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

Abstract

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.

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…However, the majority of mammalian and other eukaryotic P450s receive electrons from the diflavin enzyme NADPH-cytochrome P450 reductase in a membrane-associated class II redox system (16,17). Recent years have seen recognition of a wider diversity of redox systems driving P450 enzymes, including the well characterized Bacillus megaterium P450 BM3 (CYP102A1) fatty acid hydroxylase, a soluble P450-NADPH-cytochrome P450 reductase fusion enzyme (18), and a sterol demethylase (CYP51) class P450 fused to its cognate ferredoxin, McCYP51FX from Methylococcus capsulatus (19,20). XplA falls into a different class of P450 redox system, with a flavodoxin-like module (N-terminal) fused to the P450 (1,21,22).…”

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM‐3 heme domain

“…In this report, we address the important questions raised above using monooxygenase P450 BM-3 (CYP102A1) heme domain (BMP; amino acids Thr1-Leu455) as a model system [15,16]. P450 BM-3 is a wellstudied monooxygenase and a workhorse in directed monooxygenase evolution [1,[17][18][19][20][21][22].…”

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

“…This interaction is achieved via arginine and lysine residues on P450s and aspartic and glutamic acid residues on POR. In contrast, bacterial type 2 P450 BM3, on which the previous models were based, has an in-built flavoprotein electron transfer domain (41,42). Recent advances in crystallization and structure determination of P450 proteins has led to the availability of seven mammalian P450 structures in the PDB database, namely P450 2A6, 2B4, 2C5, 2D6, 2C8, 2C9 and 3A4.…”