Possible allostery and oligomerization of recombinant plastidial sn-glycerol-3-phosphate acyltransferase.
Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90μM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
Xue
X
Chen
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
Robin
R
Miles
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
Crystal
C
Snyder
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
Martin
M
Truksa
Plant Biotechnology, Alberta Innovates-Technology Futures, Vegreville, Alberta T9C 1T4, Canada.
Jian
J
Zhang
Plant Biotechnology, Alberta Innovates-Technology Futures, Vegreville, Alberta T9C 1T4, Canada.
Saleh
S
Shah
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. Electronic address: randall.weselake@ualberta.ca.
Randall J
RJ
Weselake
D000215:Acylation; D000494:Allosteric Regulation; D001483:Base Sequence; D060365:Chloroplast Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D032601:Erysimum / Q000201:enzymology / Q000235:genetics; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism; D007700:Kinetics; D010802:Phylogeny; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D060365:Chloroplast Proteins; D018744:DNA, Plant; D005994:Glycerophosphates; D011994:Recombinant Proteins; C029620:alpha-glycerophosphoric acid; D005992:Glycerol-3-Phosphate O-Acyltransferase
Biochemical regulation; Erysimum asperum; GPAT; Glycerolipid biosynthesis; Homotrimer; Substrate selectivity