Functional investigation of the proposed dehydratase(DH) domain of ATX, a 6-methiylsalicylic acid (6-MSA) synthase from Aspergillus terreus, revealed that the domain is not involved in dehydration of the β-hydroxy triketide intermediate tethered onthe acyl carrier proten (ACP) but catalyzes thioester hydrolysis to release the product from the ACP. Thus, we renamed this domain the thioester hydrolase (TH) domain. The intermediate bound to the TH domain of mutant H972A formed in the presence of NADPH was relased as 6-MSA by both the intact ATX and by THID (a 541 aa region containing TH domain and its downstream) protein, in trans. Furthermore, THID showed a caalytic activity to hydrolyze a model substrate, 6MSA-N-acetylcysteamine. The TH domain is the first example of a ptoduct releasing domain that is located in the middle of a multi-domain iterative type I polyketide synthase (iPKS). Moreover, it is functionally defferent from serine protease-type thioesterase domains of iPKSs.
