Biochemical and structural characterization of the tautomycetin thioesterase: analysis of a stereoselective polyketide hydrolase.

Tautomycetin (TMC) is a polyketide metabolite produced by Streptomyces sp. CK4412 and Streptomyces griseochromogenes. This intriguing molecule was previously shown to possess activated T-cell-specific immunosuppressive activity with a novel mode of pharmacological action, both in vivo and in vitro. More recent studies have also revealed promising anticancer activity in a variety of models. The biosynthesis of complex polyketide compounds in bacteria often occurs by an assembly-line type mechanism that is catalyzed by type I modular polyketide synthases (PKSs). In a key final step, the characteristic macrolactone scaffold is generated for the macrolide antibiotics erythromycin and pikromycin. This termination process is catalyzed by a thioesterase (TE) domain that is located at the carboxyterminus of the final PKS elongation module. The activity of this domain results in cleavage of the acyl chain from the adjacent ACP, followed (typically) by macrocyclization. The macrolactone is often modified further to give the final bioactive compound. The structure of TMC is highly unusual; it is one of the few known examples of a polyketide natural product that bears a terminal alkene group (Figure 1). The TMC biosynthetic gene cluster (tmc) has recently been characterized, revealing two putative type I PKSs (TmcA and TmcB), along with 16 additional gene products that are presumably involved in chain construction, tailoring, and regulation (Figure 2). Based on pathway annotation and biosynthetic principles, we hypothesized that the TMC TE catalyzes termination of chain assembly through generation of the free acid, as it contains the highly conserved sequence GxSxG and GdH motifs, as well as the Ser-His-Asp catalytic triad, characteristic of the a,b-hydrolase class of serine hydrolases. Installation of the terminal olefin is presumed to occur through decarboxylative dehydration during the post-PKS maturation of the polyketide to form the final TMC product. DNAsequence analysis of open reading frames downstream of TMC reveals several potential candidate enzymes for catalyzing this transformation: two putative decarboxylases and a dehydratase. These biosynthetic steps remain unclear, thus motivating us to elucidate the biochemical details of this process. Based on our efforts to understand chain termination and terminal alkene formation in the biosynthesis of TMC, we report herein the cloning, biochemical characterization, and the 2.0 crystal structure of the TE domain for this pathway; the first high-resolution structural analysis of a linear chainterminating TE. The TMC TE was amplified from cosmid pTMC2290 and inserted into the vector pMCSG7 (see the Supporting Information). To assess enzyme function, two short-chain enantiomerically pure TMC substrate mimics were synthesized in two steps (Scheme 1). We first evaluated the hydrolysis of model substrates 4 and 5 by the TMC TE. After overnight incubation and LCMS analysis, we found that the enzyme was greater than 350 times more active toward the (R)-isomer 4 than the (S)-isomer 5 (Figure 3). This observation was surprising, as previously characterized TEs from macrolactone-forming PKSs exhibit a high degree of substrate and stereochemical tolerance. However, this selectivity is consistent with the predicted stereochemistry of the bhydroxy group (eliminated during decarboxylative dehydration) based on sequence analysis of the preceding module 9 KR domain (see the Supporting Information, Figure S2). Mutation of the TMC TE active site Ser132 to Ala completely abrogated hydrolysis of the substrate (not shown) and confirmed its key role in catalysis. Figure 1. TMC, produced by Streptomyces sp. CK4412.