Endoplasmic oleoyl-PC desaturase references the second double bond

Area of Science:

• Biochemistry
• Plant Molecular Biology
• Enzymology

Background:

• The fad2 gene encodes the microsomal oleoyl-PC desaturase in higher plants.
• Previous understanding of plant desaturase regiospecificity was limited, focusing on carboxyl or methyl termini.
• Microsomal desaturases are crucial for introducing unsaturation into fatty acids within membrane lipids.

Purpose of the Study:

• To elucidate the precise regiospecificity of the plant oleoyl-PC desaturase (fad2 gene product).
• To determine the mechanism by which this enzyme locates and introduces a second double bond into monounsaturated fatty acid substrates.
• To validate the findings using a heterologous expression system.

Main Methods:

• Cloning and expression of peanut (Arachis hypogaea L) oleoyl-PC desaturase cDNA in yeast (Saccharomyces cerevisiae).
• Analysis of fatty acid desaturation products in yeast membrane lipids.
• Characterization of substrate specificity using endogenous and supplemented fatty acids.

Main Results:

• The expressed enzyme desaturated endogenous palmitoleate to 9(Z)12(Z)C16:2 and oleate to linoleate (9(Z)12(Z) octadecadienoate).
• Exogenously supplied cis 10-nonadecenoate was desaturated to 10(Z)13(Z)C19:2.
• The regiospecificity was best described by the Delta(x+3) rule, where x indicates the position of the existing double bond.

Conclusions:

• The plant oleoyl-PC desaturase (fad2) exhibits unique regiospecificity, referencing the existing double bond rather than the fatty acid termini.
• The Delta(x+3) rule accurately predicts the enzyme's activity on monounsaturated fatty acyl substrates.
• This finding refines our understanding of fatty acid metabolism and desaturation in plants.