PDAT, PSAT, PLA1 and ? An overview LCAT-like enzymes in plants and yeast

Abstract

A transacylation of acyl groups from phospholipids to diacylglycerols to form triacylglycerols was first characterized in microsomal preparations from developing castor beans. It was postulated that this reaction was responsible for the selective transfer of ricinoleic acid, formed on phosphatidylcholine, to the triacylglycerols (oil) in castor bean seeds. Microsomal preparations of baker’s yeast was also shown to have phospholipid:diacylglycerol acyltransferase activity. The enzyme (PDAT) responsible for this reaction was identified by an assumed homology to the animal Lechitin:Cholesterol Acyltransferase (LCAT) enzyme. Yeast contains one gene with homology to the LCAT and microsomal fractions from a knock out mutant in this gene had no PDAT activity whereas overexpressers had much increased activity. Searching the gene data bases yielded a family of six Arabidopsis genes with homology to yeast PDAT and animal LCAT. We have now identified the catalytic function of three of the enzymes encoded by these genes. The closest homolog to the yeast PDAT is a plant PDAT whereas another enzyme is catalyzing the transacylation of acylgroups from phospholipids to sterols (Phosholipid:Sterol Acyltransferase, PSAT) and a third is, when expressed in yeast, a phospholipase A1. We are currently in the process of identifying the catalytic activity of the proteins encoded by the remaining three genes. The physiological function of the PSAT enzyme in Arabidopsis seems quite clear. Data from knock out of PSAT indicate that it is the main Arabidopsis enzyme responsible for sterol ester synthesis. Although in yeast the PDAT is, together with the DGAT2, responsible for over 95% of the triacylglycerols formed in this organism, the function of its counterpart in Arabidopsis is still obscure. Knock outs of the Arabidopsis PDAT gene and its close homolog (tentatively named PDAT2) have no phenotype. Detailed biochemical investigations of the Arabidopsis PSAT enzyme showed that it is allosteric regulated by end product sterols and that the activated enzyme prefers sterol intermediates. This allosteric regulation is similar to the results very recently reported for the animal intracellular sterol ester synthesizing enzyme ACAT (Acyl-CoA:Sterol Acyltransferase), an enzyme which is evolutionary unrelated to the PSAT. In view of the results obtained regarding the allosteric regulation of PSAT we are now investigating if the evolutionary related PDAT also has such a regulation. Preliminary data of a candidate lipid regulating PDAT activity will be presented. The predicted cytosolic and membrane spanning region of the N-terminal part of the yeast (S. cerevisiae) PDAT gene was deleted and the remaining C-terminal part of the gene was expressed in Pichia pastories behind a methanol induced promoter with the -factor secretion signal in frame at the N-terminal end. The transformed Pichia secreted a soluble PDAT with high catalytic activity. This allowed us to investigate substrate specificities of the the PDAT without endogenous membrane lipids present. PDAT showed a surprisingly broad specificity towards various lipids both as acyl acceptors and donors and was also shown to have phospholipase C activity

Conference

2nd Swedish Plant Lipid Meeting

SLU Authors

• Stymne, Sten

  • Department of Crop Science, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Agricultural Science
Food Science


Permanent link to this page (URI)

https://res.slu.se/id/publ/6752