Alterations to the catalytic properties of methylketone synthase 2 from eggplant (Solanum melongena) by mutating the conserved aspartate into glutamate

Methylketone synthase 2 (MKS2) has been widely found in the plant kingdom and identified as a single-hotdog-fold acyl-lipid thioesterase (ALT) which mainly hydrolyzes the thioester bond in 3-ketoacyl-acyl carrier protein (3-ketoacyl-ACP) intermediates of the fatty acid biosynthetic pathway into free 3-keto fatty acids. Our previous study identified SmMKS2–2 as one of two functional ALTs in eggplant Solanum melongena. To gain mechanistic insights into catalysis by this enzyme, we herein combined biochemical and in silico structural analyses on SmMKS2–2. While SmMKS2–2 is capable of producing a wide range of 3-ketoacids from corresponding 3-ketoacyl-ACP substrates, SmMKS2–2-D77E mutant variant drops its thioesterase activity to the undetectable level. Consistently, the structural modelling of the D77E mutant displays that the orientation of the side chain carboxylate group of the replacing amino acid has been shifted compared to that of the native residue, resulting in smaller surface area of binding pocket that would dismiss nucleophilic catalysis of the mutant protein. Together, these data suggested that D77 is critical and specific for SmMKS2–2 to hydrolyze the thioester bond of acyl-ACP.