determining the function of hotdog-fold thioesterases

The enzymes that hydrolyze thioesters to the free acids and free thiols are called thioesterases. Based on the different folding strategy, most thioesterases are classified into two superfamiles: the hotdog-fold enzyme superfamily or alpha/beta-fold hydrolase enzyme superfamily. Hotdog-fold thioesterases share high degree similarity in structure which resembles a “hotdog”. YigI is one of the last two uncharacterized hotdog-fold thioesterases from E.coli. To study the function of YigI, substrate specificity profile was determined by thioesterase activity assay. YigI possesses a preference for long chain acyl-CoA over shorter chain acyl-CoA. The highest activity was found in catalysis of myristoyl-CoA. By alignment with the closest homolog, the uncharacterized tetrameric type AB thioesterase from Shewanella oneidensis, the important catalytic residues of YigI were predicted as following: Asp69, Gln56 and His60. Enzyme activity decreased after the site-direct mutagenesis of those residues.