(132g) Phylogenetic and Experimental Characterization of An Acyl-ACP Thioesterase Family Reveals Significant Diversity In Enzymatic Specificity and Activity
Acyl-ACP thioesterases (TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl-chain elongation of fatty acid biosynthesis. It is also the biochemical determinant of the fatty acid compositions of storage lipids in plant seeds. To explore enzymatic diversity and to identify novel TEs, product distributions and activities of 31 TEs in an acyl-ACP TE family were determined. These TEs were chosen by two different approaches: 1) selected from public databases on the basis of a phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) molecularly cloned organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of all the TEs were determined in E. coli. Based on their specificities, these TEs were clustered into classes: 1) Class I TEs that act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II TEs that have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III TEs that act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs produced short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.