Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana.
Towards defining the function of Arabidopsis thaliana fatty acid hydroxylases, five members of the CYP86A subfamily have been heterologously expressed in baculovirus-infected Sf9 cells and tested for their ability to bind a range of fatty acids including unsubstituted (lauric acid (C12:0) and oleic acid (C18:1)) and oxygenated (9,10-epoxystearic acid and 9,10-dihydroxystearic acid). Comparison between these five P450s at constant P450 content over a range of concentrations for individual fatty acids indicates that binding of different fatty acids to CYP86A2 always results in a higher proportion of high spin state heme than binding titrations conducted with CYP86A1 or CYP86A4. In comparison to these three, CYP86A7 and CYP86A8 produce extremely low proportions of high spin state heme even with the most effectively bound fatty acids. In addition to their previously demonstrated lauric acid hydroxylase activities, all CYP86A proteins are capable of hydroxylating oleic acid but not oxygenated 9,10-epoxystearic acid. Homology models have been built for these five enzymes that metabolize unsubstituted fatty acids and sometimes bind oxygenated fatty acids. Comparison of the substrate binding modes and predicted substrate access channels indicate that all use channel pw2a consistent with the crystal structures and models of other fatty acid-metabolizing P450s in bacteria and mammals. Among these P450s, those that bind internally oxygenated fatty acids contain polar residues in their substrate binding cavity that help stabilize these charged/polar groups within their largely hydrophobic catalytic site.
Rupasinghe
Sangeewa G
SG
Department of Cell and Developmental Biology, University of Illinois, Urbana, Illinois 61801, USA.
Duan
Hui
H
Schuler
Mary A
MA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D016415:Sequence Alignment; D017385:Sequence Homology
D029681:Arabidopsis Proteins; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C111056:CYP86A1 protein, Arabidopsis