Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

Abstract

Structural control of chemoselectivity, stereoselectivity, and substrate specificity in membrane-bound fatty acid acetylenases and desaturases Gagné, Steve J.; Reed, Darwin W.; Gray, Gordon R.; Covello, Patrick S. Saskatoon SK S7N 5A8, Canada Received September 14, 2009; Revised Manuscript Received October 26, 2009 ABSTRACT: The FAD2-like desaturases comprise a group of membrane-bound oxygenases involved in the modification of fatty acyl groups in plants and fungi. This group includes typical oleate desaturases which introduce a Δ12 cis double bond and more unusual enzymes such as Crep1, an acetylenase from the plant Crepis alpina, which introduces a triple bond in linoleate at the Δ12 position. In this study, the structure--function relationship between FAD2-like acetylenases and desaturases was examined through site-directed mutagenesis and heterologous expression. Eleven amino acid positions were identified that show complete evolutionary conservation within acetylenases or desaturases but have different amino acids in the other class of enzyme. Point mutants in Crep1 were constructed and expressed in yeast to test the role in fatty acid modification of the amino acids at the 11 positions. Results indicate the importance of five amino acid positions within Crep1 with regard to desaturase and acetylenase chemoselectivity, stereoselectivity, and substrate recognition. For example, relative to wild-type Crep1, the Y150F, F259L, and H266Q mutations all favored desaturation over acetylenation. The data indicate that small changes in primary sequence, particularly in the vicinity of the active site, can have profound changes on chemoselectivity and other aspects of the function of membrane-bound desaturase-like enzymes.The FAD2 1 -like desaturases comprise a group of membranebound oxygenases involved in the modification of fatty acyl groups in plants and fungi. The archetypal plant FAD2 catalyzes the introduction of a cis double bond at the Δ12 position of oleoylphosphatidylcholine in the endoplasmic reticulum (1) (Figure 1). This requires molecular oxygen and reducing equivalents from cytochrome b 5 (2).Interestingly, the FAD2-like enzyme group includes a number of variants which catalyze reactions other than the usual Δ12 cis desaturation. These reactions include hydroxylation, epoxidation, conjugated double bond formation, and triple bond formation (3). The acetylenase Crep1, from Crepis alpina, is the first of a number of FAD2-like acetylenases identified in the plant families Asteraceae, Apiaceae, and Araliaceae (4). Crep1 catalyzes the formation of a triple bond at the 12,13 position of linoleate (18:2-9c,12c) to give crepenynate (18:1-9c,12a) (Figure 1). Crep1 has also been reported to catalyze both cis and trans desaturation of oleate (5) (Figure 1).In general, the membrane-bound desaturases are particularly difficult to work with in vitro.Muchofwhatisknownaboutthe structure and function of these enzymes has been derived from their primary structure and in vivo activities in yeast. By analogy to solub...

Mechanistic and Structural Insights into the Regioselectivity of an Acyl-CoA Fatty Acid Desaturase via Directed Molecular Evolution

“…Two mutations, AdD12/9-A98V and AdD12/9-K136R, were found to be located within or close to the first two conserved histidine boxes that together with the third histidine box are believed to form the active site (44). Residues in the vicinity of the histidine boxes and particularly in the amino acid stretch following the second histidine box have been reported to influence the catalytic reaction outcome as well as the chain length specificity of FAD2-related enzymes such as the Lesquerella fendleri ⌬12-hydroxylase and the Crepis alpina ⌬12-acetylenase (45)(46)(47). Among the cricket ⌬12/⌬9-desaturase single mutants, AdD12/ 9-K136R displayed the largest increase in desaturase activity.…”

De novo biosynthesis of terminal alkyne-labeled natural products

“…Terminal alkynes can be formed by acetylenases, a special family of desaturases that catalyze O 2 -dependent dehydrogenation of C-C bonds in a diiron dependent mechanism 12 . Several membrane-bound acetylenases have recently been 3 identified from plants, insects, fungi, and bacteria 4,10,[13][14][15][16][17] , but all of these reports have been limited to bioinformatics or in vivo studies through mutagenesis or heterologous expression in yeasts and plants because it is notoriously difficult to work with the membrane-bound desaturases in vitro. As a result, no definitive information on the reactions catalyzed by these enzymes is available to date.…”

Conjugated Fatty Acid Synthesis

“…, the FAD2-like desaturase product; and C. alpina acetylenase preferentially produces 18:2 ⌬ 9cis,12trans (10,40,41). This suggests that in the case of tung conjugase and C. alpina acetylenase some additional changes near the active site may be necessary for introducing preferential FAD2-like desaturase activity.…”