Oxygenation of polyunsaturated long chain fatty acids by recombinant CYP4F8 and CYP4F12 and catalytic importance of Tyr-125 and Gly-328 of CYP4F8.
Recombinant CYP4F8 and CYP4F12 metabolize prostaglandin H2 (PGH2) analogs by omega2- and omega3-hydroxylation and arachidonic acid (20:4n-6) by omega3-hydroxylation. CYP4F8 was found to catalyze epoxidation of docosahexaenoic acid (22:6n-3) and docosapentaenoic acid (22:5n-3) and omega3-hydroxylation of 22:5n-6. CYP4F12 oxidized 22:6n-3 and 22:5n-3 in the same way, but 22:5n-6 was a poor substrate. The products were identified by liquid chromatography-mass spectrometry. The missense mutation 374A>T of CYP4F8 (Tyr125Phe in substrate recognition site-1 (SRS-1)) occurs in low frequency. This variant oxidized two PGH2 analogs, U-51605 and U-44069, in analogy with CYP4F8, but 20:4n-6 and 22:5n-6 were not oxidized. CYP4F enzymes with omega-hydroxylase activity contain a heme-binding Glu residue, whereas CYP4F8 (and CYP4F12) with omega2- and omega 3-hydroxylase activities has a Gly residue in this position of SRS-4. The mutant CYP4F8 Gly328Glu oxidized U-51605 and U-44069 as recombinant CYP4F8, but the hydroxylation of arachidonic acid was shifted from C-18 to C-19. Single amino acid substitutions in SRS-1 and SRS-4 of CYP4F8 may thus influence oxygenation of certain substrates. We conclude that CYP4F8 and CYP4F12 catalyze epoxidation of 22:6n-3 and 22:5n-3, and CYP4F8 omega3-hydroxylation of 22:5n-6.
Stark
Katarina
K
Department of Pharmaceutical Biosciences, Division of Biochemical Pharmacology, Uppsala University, SE-751 24 Uppsala, Sweden.
Wongsud
Buanus
B
Burman
Robert
R
Oliw
Ernst H
EH
D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000737:chemistry* / Q000235:genetics; D005998:Glycine / Q000737:chemistry* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D014443:Tyrosine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C422180:CYP4F12 protein, human; C120451:CYP4F8 protein, human; D010100:Oxygen; D005998:Glycine