Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.
The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.
Fairhead
Michael
M
Division of Molecular Biosciences, Faculty of Life Sciences, Imperial College London, South Kensington, SW7 2AZ, London, UK.
Giannini
Silva
S
Gillam
Elizabeth M J
EM
Gilardi
Gianfranco
G
D001711:Biotransformation; D002753:Chlorzoxazone / Q000737:chemistry; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000235:genetics*; D015786:Cytochromes b5 / Q000737:chemistry; D006801:Humans; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D009596:Nitrophenols / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D009596:Nitrophenols; D011993:Recombinant Fusion Proteins; D009249:NADP; D015786:Cytochromes b5; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone; D010100:Oxygen; C024836:4-nitrophenol