Trans-acyltransferase (trans-AT) polyketide synthase (PKS) is a variant form of the multienzyme modular type I PKS. About 38% of the PKS genes predicted by genomic data analysis are estimated to be trans-AT PKS. Significant portions of the predicted gene cluster are expected to produce previously unknown compounds. Condensation-incompetent ketosynthase domains (KS0) are a type of ketosynthase domain ubiquitously found in trans-AT PKSs. Their lack of decarboxylative condensation activity is due to the mutation of a conserved His residue. KS0s are found in various biosynthetic pathways of trans-AT PKSs, in front of various enzymes such as enoylreductase, ketosynthase, O-methyltransferase, methyltransferase, and thioesterase. The locations are diverse, they can be found within modules, at the end of split modules and trans-PKS-NPRS junctions. The role and mechanism of KS0 is not well understood. In our laboratory, the activity and the mechanism of KS0 from Difficidin biosynthase (DfnKS05) is being studied. Peptide mass spectrometry data showed the DfnKS05 can accept various types of carbon substrates. It also catalyzed translocation of substrates between different ACPs. The peptide mass spectrometry and enzyme kinetics data revealed that ACPs are competitively modified either by trans-AT or by KS0. KS0 determines the fate of ACP modification by inhibiting the trans-AT activity. Enzyme kinetic data showed that the inhibition mechanism of KS0 resembles a competitive inhibitor. This may imply that there is direct protein-protein interaction between ACP and KS0. The sequence-function relationship of KS0 is not well known. A reverse mutant, N1934H DfnKS05, was generated in order to test the role of mutated residue in KS0s. We hypothesized that N1934H DfnKS05 would recover the condensation activity of ketosynthase since the conserved His residue was restored. Unexpectedly, N1934H DfnKS05 did not regain the condensation activity but the translocation activity was retained. This implies that the single residue does not determine the activity of KS0. Further study is needed to complete the analysis of sequence / function activity.

Activity of KS0 in trans-AT PKS biosynthase: Control of the ACP modification by inhibition of acyltransferase

254th National Meeting (2017)

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