Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119

Laura S. Koo, Chad E. Immoos, Michael S. Cohen, Patrick J. Farmer, Paul R. Ortiz de Montellano

Research output: Contribution to journalArticle

85 Scopus citations

Abstract

CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450cam can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.

Original languageEnglish (US)
Pages (from-to)5684-5691
Number of pages8
JournalJournal of the American Chemical Society
Volume124
Issue number20
DOIs
StatePublished - May 22 2002

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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