Article Figures & Data
Figures
- Fig. 1.
Characterization of CYP4Z1 expressed in yeast microsomes. (A) Western blot of microsomes harvested from untransformed yeast (lane 1) and from yeast expressing CYP4Z1 (lane 2). (B) Carbon monoxide difference spectrum of reduced CYP4Z1 microsomes.
- Fig. 2.
GC-MS chromatogram of LA metabolites generated by recombinant CYP4Z1. The chromatograms show resolution of the hydroxylated products generated from incubations of LA with CYP4Z1-expressing yeast microsomes in the presence (bottom traces) and absence (top traces) of NADPH cofactor (LA metabolites were quantitatively converted to their (bis)trimethylsilyl derivatives before analysis). Individual metabolites were identified by selected ion monitoring (SIM) of the optimal, differentiating mass fragmentation ions for each derivatized regioisomer (7-OH LA, m/z = 173; 8-OH LA, m/z = 159; 9-OH LA, m/z = 145; 10-OH LA, m/z = 131; 11-OH LA, m/z = 117 and 12-OH LA, m/z = 345). 12-OH LA (Rt = 15.6 minutes) concentrations were below the limit of detection (i.e., <0.02 pmol were produced/min per picomoles CYP4Z1).
- Fig. 3.
HET0016 inhibition of CYP4Z1-catalyzed LA metabolism. The overlaid graphs show the relative IC50 curves for inhibition of yeast microsomal CYP4Z1-mediated LA 8-, 9-, and 10-hydroxylase activity by HET0016, a low nanomolar inhibitor of most CYP4 enzymes. Data points denote the mean turnover rate of LA to each metabolite, obtained from duplicate incubations of 100 μM LA and HET0016 (at concentrations ranging from 0.1 to 500 μM) with CYP4Z1 microsomes, expressed as a percentage of the rate obtained in the absence of inhibitor. HET0016 inhibited formation of 8-OH LA, 9-OH LA, and 10-OH LA with IC50 values of 14.7, 14.9, and 15.9 μM, respectively (we were unable to generate reasonable IC50 curves in this experiment for either 7-OH LA, owing to low turnover rates, or 11-OH LA, owing to overlap of the derivatized metabolite peak with HET0016).
- Fig. 4.
ESI− LC-MS/MS chromatograms of AA metabolism by cytochrome P450s. Total ion chromatograms (TICs) of metabolites produced from incubations of 75 μM AA with CYP2C19 and CYP4F2 Supersomes and with CYP4Z1 yeast microsomes, in the presence and absence of NADPH (TICs = sum of 17 individual AA metabolite MRM channels plus channels for the internal standards, 20-HETE-d6 and 14,15-EET-d11). An additional TIC is included to show (the lack of) HET0016 inhibition of CYP4Z1 AA metabolism at 1 μM HET0016. Insets represent magnifications of individual MRM chromatograms from the (+) NADPH incubations of P450 with AA (peaks are normalized to the same scale): (A) m/z = 325 > 281 (20-HETE-d6), (B) m/z = 333 > 271 (20-COOH-AA), (C) m/z = 319 > 245 (20-HETE), and (D) m/z = 319 > 275 (19-HETE).
- Fig. 5.
LC-MS/MS chromatograms showing stereoselective formation of 14,15-EET by CYP4Z1 and CYP2C19. The chromatograms show chiral resolution of the 14,15-EET metabolic products isolated from incubations of AA with either CYP4Z1 yeast microsomes or CYP2C19 supersomes (with (±)14,15-EET-d11 added as internal standard). Metabolites and standards were derivatized to their PFB esters and then analyzed by electron-capture APCI− LC-MS/MS (resolution was achieved on a Chiralpak AD column).
- Fig. 6.
3D Homology models of the CYP4Z1 active site with LA and AA docked. The CYP4Z1 amino acid residues shown in light blue are believed to be of primary importance to substrate binding, with Leu117, Val126, and Val379 forming a hydrophobic pocket encapsulating the fatty chains of LA (green, A) and AA (lavender, B). Asn381 appears to anchor both substrates in the active site, likely through electrostatic interactions formed between the Asn amine and the fatty acid carboxylate moieties (∼2 Å distant). (A) The model of LA bound to CYP4Z1 shows internal carbons of the fatty acid centered over the heme (brown), with C-8 through C-11 of LA all located between 3.7 and 5.0 Å from the heme iron atom. By contrast, the terminal carbon of LA is oriented away from the heme iron at 6.3 Å. (B) The model of AA bound to CYP4Z1 likewise shows the terminal carbon of the fatty acid to be pointed away from the heme, whereas the C14-C15 double bond of AA is only 5 to 6 Å distant from the heme iron. In the figure, oxygen atoms are shown in red, nitrogen atoms in dark blue, sulfur atoms in yellow, and the heme iron as a brown sphere.
Tables
- TABLE 2
Relationship between the presence of a predicted heme covalent link and 20-HETE formation in human CYP4 enzymes
Subfamily Enzyme Localization Covalent Heme AA Metabolite CYP4A 4A11 Liver, kidney Yes (FEGHDT) 20-HETE Powell et al. (1998) 4A22 Liver, kidney Yes (FEGHDT) — CYP4FB 4B1 Lung, bladder Yes (FEGHDT) — CYP4V 4V2 Retina Yes (FEGHDT) 20-HETE Nakano (2011) CYP4F 4F2 Liver, kidney Yes (FEGHDT) 20-HETE Powell et al. (1998) 4F3A Myeloid tissues Yes (FEGHDT) 20-HETE Fer et al. (2008) 4F3B Liver, kidney Yes (FEGHDT) 20-HETE Christmas et al. (2001) 4F11 Liver, kidney Yes (FEGHDT) 20-HETE Tang et al. (2010) 4F22 Skin Yes (FEGHDT) — 4F8 Urogenital No (FGGHDT) 18-HETE Bylund et al. (2000) 4F12 Liver, intestine No (FGGHDT) 18-HETE Bylund et al. (2001) CYP4X 4X1 Brain, skin No (LAGHDT) 14,15-EET Stark et al. (2008) CYP4Z 4Z1 Breast tissue No (FAGHDT) 20-HETE Yu et al. (2012) 4Z1 Breast tissue No (FAGHDT) 14,15-EET (current study)
Data Supplement
- Supplemental Data -
Supplemental Table 1 - Primers used for vector construction
Supplemental Figure 1 - Plasmid sequences
Supplemental Figure 2 - ESI- LC-MS/MS chromatograms showing AA metabolism by CYP4F12 Supersomes
Supplemental Figure 3 - HET0016 inhibition of CYP4F12-catalyzed AA metabolism
- Supplemental Data -
