Human cytochrome P450 4F11: Heterologous expression in bacteria, purification, and characterization of catalytic function

Abstract

Human cytochrome P450 (P450) 4F11 is still considered an "orphan" because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing ~230 nmol P450 from a 3-liter culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Z. Tang et al., Anal. Chem. 81, 3071-3078, 2009). Four fatty acids-palmitic, oleic, arachidonic, and docosahexaenoic-were identified in human liver and verified as substrates of P450 4F11. The products were characterized as ω-hydroxylated fatty acids by gas chromatography-mass spectrometry analysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug Ndemethylation reactions but none for activation of several procarcinogens.

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

“…The kinetic parameters k cat and K m were estimated based on MichaelisMenten plots and nonlinear regression analysis ( Table 2 ). The catalytic effi ciencies ( k cat / K m ) of P450 2W1-catalyzed fatty acid oxidations were comparable with those catalyzed by other human P450 enzymes ( 19,21 ). The catalytic efficiency of 18:1 LPC oxidation was ‫ف‬ 6-fold greater than that of the FFA C18:1.…”

“…FFAs are common substrates for a number of human and other P450 enzymes ( 12,21,(41)(42)(43). Among the fi ve fatty acids identifi ed in our study, only C20:4 has been reported as a substrate for P450 2W1 previously ( 12 ).…”

“…2 ). Although many other human P450 enzymes share similar catalytic effi ciencies toward fatty acids ( 19,21 ), they showed little activity with 18:1 LPC. We conclude that 18:1 LPC is a substrate relatively specifi c for P450 2W1.…”

“…Incubation of a 1:1 mixture of 18 O-and 16 O-labeled oxygen gas with tissue extracts generates products as M/M+2 doublets in the MS spectra, which can be identifi ed by the program DoGEX ( 19,20 ). This strategy has been validated ( 19 ) and used to identify endogenous substrates for P450 4F11 (i.e., FFAs) ( 21 ).…”

“…The purifi ed enzyme was used to conduct untargeted substrate searches in human co lorectal cancer samples (i.e., site of P450 2W1 expression) using the LC/MS metabolomic and isotopic labeling approach ( 19,21 ). A series of lysophospholipids and FFAs were identifi ed as novel substrates for P450 2W1, and the isomerand enentiomer-selectivity of P450 2W1-catalyzed lysophospholipid oxidations have been characterized.…”