Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Edgar B. Cahoon, Ylva Lindqvist, Gunter Schneider, John Shanklin

Research output: Contribution to journalArticle

129 Scopus citations

Abstract

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl- ACP desaturases. By replacement of specific amino acid residues in a Δ6- palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ6 or Δ9 positions of 16:0-and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ9- 18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P1791). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

Original languageEnglish (US)
Pages (from-to)4872-4877
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number10
DOIs
StatePublished - May 13 1997

Keywords

  • diiron
  • nonheme iron
  • protein engineering
  • rational design
  • unsaturated fatty acid

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position'. Together they form a unique fingerprint.

  • Cite this