Substrate specificity of an archaeal thioesterase from Thermococcus kodakarensis

Thioesterases catalyze the hydrolysis of thioester bonds and are responsible for reactions such as the release of fatty acids from acyl-ACP in fatty acid biosynthesis. Although enzymes from bacteria and eukaryotes have been studied, identification and characterization of thioesterases from archaea have not been reported. Here we searched the genome of the hyperthermophilic archaeon Thermococcus kodakarensis for candidate genes that might encode a thioesterase. Among these, the TK1652 protein contained a HotDog fold and shared ~ 20% identity with TE8 and TE13 family thioesterases. Active site residues proposed for TE8 and TE13 enzymes were partially conserved in the TK1652 protein. The recombinant TK1652 protein produced in Escherichia coli was purified to apparent homogeneity. Although TE8 and TE13 family enzymes assemble as tetramers, the TK1652 protein formed a homodimer. The protein displayed thioesterase activity toward acetyl-CoA, and to a much lower extent, propionyl-CoA. The TK1652 protein did not recognize phenylacetyl-CoA, n-decanoyl-CoA, lauroyl-CoA and myristoyl-CoA, substrates utilized by TE8/TE13 family thioesterases. Differences in active site residues, quaternary structure, and substrate specificity suggested that the TK1652 protein represents a new family of thioesterases. Acetyl-CoA thioesterase activity was detected in cell-free extracts of T. kodakarensis, and the metabolic roles of this activity are discussed.