Synthesis And Reactivity Of Different Acyl-ACP Mutants

Triglyceride(TAG)synthesized in the biological pathways is an important bioenergy precursor.Acyl-acyl carrier protein(acyl-ACP),as an important substrate in its synthetic pathway,catalyzed by glycerol-3-phosphate acyltransferase(GPAT)with glycerol-3-phosphate(G-3-P)to synthetize lysophosphatidic acid(LPA)is the first reaction and plays an important regulatory role in the synthesis of TAG.Therefore,the study on the reaction of acyl-ACP with GPAT is of great significance.When ACP is involved as a part of the acyl-ACP substrate in the GPAT catalyzed acyl transfer reaction,it is usually only considered as the catalytic selectivity of the acyl chain to GPAT,ignoring the contribution of ACP in it.This selection mechanism is misunderstood in many enzymes that catalyze ACP reactions.Therefore,in order to study the selective contribution of ACP fraction of acyl-ACP to GPAT,obtaining different chain lengths of acyl-ACP and its mutants,and analyzing its reaction characteristics with GPAT is an important prerequisite for understanding the structure and function of ACP and understanding the molecular mechanism of ACP involved in enzyme reaction kinetics.In this study,based on the amino acid sequence and three-dimensional structure information of wild-type ACP,a total of 12 ACP mutants involving 11 amino acid sites were designed.The expression plasmids of 12 ACP mutants were constructed in vitro and expressed in E.coli.The active ACP(holo-ACP)bound to the 4'-phosphopantetheinyl group(4'-PPT)was reacted with 16:0,18:1 free fatty acids under the action of acyl-ACP synthetase to synthesize 16:0-ACP and 18:1-ACP.Then 16:0-ACP and 18:1-ACP were purified by size exclusion chromatography.Finally,the enzyme activity of 16:0-ACP and 18:1-ACP and its mutants with GPAT were measured,and the reactivity was analyzed.The results showed that 12 ACP mutants were expressed and purified.The expresssion of the target protein,the elution volume in size exclusion chromatography and the purity of holo-ACP in high performance liquid chromatography(HPLC)were different.The 16:0-ACP and 18:1-ACP synthesized products were purified and qualitatively and quantitatively analyzed by HPLC.The results showed that the stability of acyl-ACP and its mutants with different chain lengths prominently changed.In addition,in the enzyme activity assay with GPAT,compared with WT,16:0-ACP and 18:1-ACP after site-directed mutagenesis have undergone tremendous changes in the reaction characteristics with GPAT,and the preference of GPAT for chain length has also changed.In summary,the study of the reactivity of 16:0-ACP and 18:1-ACP and their mutants with GPAT laid the foundation for the structure and function of ACP and the interaction mechanism with related enzymes.