Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana

Sangeewa G. Rupasinghe, Hui Duan, Mary A Schuler

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)279-293
Number of pages15
JournalProteins: Structure, Function and Genetics
Volume68
Issue number1
DOIs
StatePublished - Jul 1 2007

Fingerprint

Mixed Function Oxygenases
Arabidopsis
Fatty Acids
lauric acid
Oleic Acid
Heme
Substrates
Cytochrome P-450 CYP4A
Sf9 Cells
Mammals
Baculoviridae
Titration
Catalytic Domain
Bacteria
Crystal structure
Enzymes

Keywords

  • Arabidopsis
  • Cytochrome P450 monooxygenases
  • Fatty acid hydroxylases
  • Molecular modeling
  • Substrate-docking
  • Substrate-protein interactions

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana. / Rupasinghe, Sangeewa G.; Duan, Hui; Schuler, Mary A.

In: Proteins: Structure, Function and Genetics, Vol. 68, No. 1, 01.07.2007, p. 279-293.

Research output: Contribution to journalArticle

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