Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.
The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.
Dodhia
Vikash Rajnikant
VR
Division of Molecular Biosciences, Imperial College London, Biochemistry Building, South Kensington, London, SW7 2AY, UK.
Fantuzzi
Andrea
A
Gilardi
Gianfranco
G
D020816:Amino Acid Motifs; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D002384:Catalysis; D002942:Circular Dichroism; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004917:Erythromycin / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009249:NADP; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases