Structural control of chemoselectivity, stereoselectivity, and substrate specificity in membrane-bound fatty acid acetylenases and desaturases.
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 Delta12 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 Delta12 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.
Gagné
Steve J
SJ
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon SK S7N 0W9, Canada.
Reed
Darwin W
DW
Gray
Gordon R
GR
Covello
Patrick S
PS
D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D027468:Crepis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D005227:Fatty Acids; D010940:Plant Proteins; D044943:Fatty Acid Desaturases