A Model of Structure and Catalysis for Ketoreductase Domains in Modular Polyketide Synthases

Abstract

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.

The ‘gifted’ actinomycete Streptomyces leeuwenhoekii

“…Reductase-active KR domains can be inferred by the presence of three diagnostic regions for binding NADPH: a GxGxxG motif, a swap linker and a WGxW motif (Garg et al 2014). Also the presence of the catalytic triad lysine, serine and tyrosine should also be present in active KR domains (Reid et al 2003). All of the KR domains found in the chaxalactin PKS possess all of the described motifs and are expected to be active, consistent with the structures of chaxalactins A-C.…”

“…Thus the export of this lasso peptide is presumably mediated by a non-specific efflux pump present in many streptomycetes (Gomez-Escribano et al, unpublished results). The amino acid sequence of the predicted mature form of the lasso peptide of lp2 appears thus far to be novel and could not be detected in the draft genome sequence of strain C58 (GomezEscribano et al 2015;Razmilic 2017); lp2 is currently under study (Gomez-Escribano et al, unpublished results).…”

The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

“…Interestingly, both of these split modules may introduce an unusual cis olefin into the growing chain. Earlier work on the stereochemistry of the KR reaction in polyketide biosynthesis linked the presence/absence of a conserved aspartate residue in the KR domain with the stereochemical outcome of the subsequent dehydration reaction (16)(17)(18). Indeed, the three modules in the PksX synthase that generate a cis double bond lack this conserved aspartate in the KR domain (i.e., PksJ-KR2, PksL-KR2, and PksM-KR1).…”

The Complete Biosynthetic Gene Cluster of the 28-Membered Polyketide Macrolactones, Halstoctacosanolides, from Streptomyces halstedii HC34

“…However, since all modules contain KR domain, certain contradiction appeared between the gene structure and the chemical structure. Some inactive KR domains have been reported to lack the active site motif, GXGXXGXXXA for NADP-binding or the catalytic triad, Lys, Ser and Tyr in the active site [8]. However, in the case of hls KR, all the important amino acid residues appeared to be conserved in all KR.…”

“…Usually, most of the double bond geometry found in polyketide chains is E-configuration. It has been suggested that the stereochemistry of b-hydroxyl group derived from KR domain might control the double bond geometry in DH domain [8]. An Asp residue of KR domain was proposed to be an important residue for determining the stereospecificity.…”