Identification and Functional Characterization of a Soybean (Glycine max) Thioesterase that Acts on Intermediates of Fatty Acid Biosynthesis.

(1) Background: Plants possess many acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) with unique specificity. One such TE is methylketone synthase 2 (MKS2), an enzyme with a single-hotdog-fold structure found in several tomato species that hydrolyzes 3-ketoacyl-ACPs to give free 3-ketoacids. (2) Methods: In this study, we identified and characterized a tomato MKS2 homolog gene, namely, GmMKS2, in the genome of soybean (Glycine max). (3) Results: GmMKS2 underwent alternative splicing to produce three alternative transcripts, but only one encodes a protein with thioesterase activity when recombinantly expressed in Escherichia coli. Heterologous expression of the main transcript of GmMKS2, GmMKS2-X2, in E. coli generated various types of fatty acids, including 3-ketoacids-with 3-ketotetradecenoic acid (14:1) being the most abundant-cis-Δ5-dodecanoic acid, and 3-hydroxyacids, suggesting that GmMKS2 acts as an acyl-ACP thioesterase. In plants, the GmMKS2-X2 transcript level was found to be higher in the roots compared to other examined organs. In silico analysis revealed that there is a substantial enrichment of putative cis-regulatory elements related to disease-resistance responses and abiotic stress responses in the promoter of this gene. (4) Conclusions: GmMKS2 showed broad substrate specificities toward a wide range of acyl-ACPs that varied in terms of chain length, oxidation state, and saturation degree. Our results suggest that GmMKS2 might have a stress-related physiological function in G. max.