Characterization of the Catalytic Mechanism of AT1g78690p Acyltransferase Activity
Saparja Nag ’17 and Teresa Garrett (Chemistry)
Headgroup-acylated glycerophospholipids make up a small but significant and understudied class of lipids. AT1g78690p, an enzyme native to plant species Arabidopsis thaliana, is a peripheral membrane protein that when expressed in E. coli results in the accumulation of acylphosphatidylglycerol (acyl-PG). However, in vitro the enzyme catalyzes the acylation of mono-acylated glycerophospholipids, 1-acyl lyso PE or 1-acyl lyso PG, to form di-acylated glycerophospholipid products, PE or PG, respectively. In order to characterize the catalytic and kinetic properties of AT1g78680p, five mutant genes were constructed to heterologously overexpress the protein in E. coli. Each mutant contained a single amino acid mutation to alanine at distinct positions deemed potentially catalytically significant. The five mutated amino acids studied – threonine-63, asparagine-66, histidine-67, aspartate-72, and arginine-92 - were chosen because they were 100% conserved in sequence alignments of lysophospholipid acyltransferase (LPLAT) superfamily homologs from different species. Preliminary findings showed decreased activity in vitro for all five mutants from the wildtype AT1g78690p protein, suggesting some catalytic significance to the amino acids studied. Further testing will be conducted to determine if the mechanism relies on substrate affinity or the enzyme’s catalytic activity, as well as substrate specificity of the enzyme.