The role of thioesterase domain catalysed hydrolysis in preventing chain extension in polyketide synthases Fang Pang 1 , Marianne Costa 1 , Matthew Jenner¹, Xinyun Jian 1,2 Munro Passmore 1 , Christopher D. Fage 1 , Lona M. Alkhalaf* 1 and Gregory L. Challis* 1,2,3 1 Department of Chemistry, University of Warwick, UK, 2 Warwick Integrative Synthetic Biology Centre, University of Warwick, UK, 3 Monash University, Australia Gladiolin is a novel macrolide antibiotic with promising activity against multi-drug resistant Mycobacterium tuberculosis , isolated from Burkholderia gladioli BCC0238. 1,2 The gladiolin trans -AT polyketide synthase (PKS), responsible for the biosynthesis of gladiolin, contains an extra ketosynthase (KS) domain in its final module. Interestingly, the active site His residue in the HSVGS motif, responsible for initiating chain extension and commonly mutated in previously characterised non-elongating KS domains, is present. Here, we show the KS domain does not catalyse chain extension in vivo but that it is required for chain transacylation. Furthermore, biochemical assays showed the terminal thioesterase (TE) domain very efficiently hydrolyzes malonyl thioesters from its upstream ACP domain. Thus, the TE domain removes the malonyl thioesters from the ACP domain before chain elongation can occur. Through a combination of X-ray crystallography, phylogenetic analysis and site- directed mutagenesis, we found a key residue in the substrate pocket involved in the substrate specificity of the TE domain and show this is a universal mechanism for chain release in trans PKSs. References 1. Song, Lijiang, et al. "Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis." Journal of the American Chemical Society 139.23 (2017): 7974-7981. 2. Perry, Christopher, et al. "Relative stereochemical assignment of C-33 and C-35 in the antibiotic gladiolin." Tetrahedron 74.38 (2018): 5150-5155.
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