title: Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris.
abstract: alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.
authors.affiliation: Laboratoire de Chimie des Biomolécules et des Systèmes Organisés, CNRS UMR 6052, Ecole Nationale Superieure de Chimie de Rennes, Ave du Gal Leclerc, 35700 Rennes Beaulieu, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Daligault
authors.forename: Darwin W
authors.initials: DW
authors.lastname: Reed
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Savile
authors.forename: Caroline
authors.initials: C
authors.lastname: Nugier-Chauvin
authors.forename: Henri
authors.initials: H
authors.lastname: Patin
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Covello
authors.forename: Peter H
authors.initials: PH
authors.lastname: Buist
mesh_terms: D002708:Chlorella / Q000378:metabolism*; D015394:Molecular Structure; D017962:alpha-Linolenic Acid / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D017962:alpha-Linolenic Acid