Membrane integration of recombinant human P450 forms.
Amino terminal sequence modification of cytochrome P450 enzymes is often necessary to achieve expression in bacteria. The aim of this study was to examine the effect of such modifications on membrane integration and P450 activity. Forms that retained substantial N-terminal hydrophobic sequences remained unaffected by treatments to remove peripheral membrane proteins and were released only by detergent. Truncated P450s 2A13, 2C9 (delta 3-20), 2C19 (delta 3-20), 2D6 (DB11) and 2E1 remained principally membrane-bound, but some P450 was found in the soluble fraction and could be partially extracted by alkaline and high salt treatments. The subcellular localization of P450s 2C9 and 2C19 assessed by fluorescence microscopy mirrored the distribution between subcellular fractions. The MALLLAVFL modified forms of P450 2C9 YFP, P450 2C18 YFP and P450 2C19 YFP were found primarily at the periphery of the cells, whereas the truncated forms of P450 2C9 (delta 3-20) YFP and 2C19 (delta 3-20) YFP were observed at the periphery as well as inside the cells. N-terminal variants of P450s 2C9 and 2C19 showed altered kinetics towards form-selective substrates. Rates of diclofenac 4 -hydroxylation by P450 2C9 and luciferin H-EGE metabolism by P450 2C19 were higher for the MALLLAVFL-modified forms compared with the (delta 3-20) truncated forms despite supplementation of truncated form incubations with additional reductase. Thus, N-terminal sequence modifications changed the degree of membrane integration, potentially affecting subcellular localization, interactions with redox partners, and hence enzymatic activity.
Shukla
A
A
School of Biomedical Sciences, The University of Queensland, Brisbane, Australia. alkashukla@gmail.com
Huang
W
W
Depaz
I M
IM
Gillam
E M J
EM
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology*; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19