Amino acid-accepting ketosynthase domain from a trans-AT polyketide synthase exhibits high selectivity for predicted intermediate

Abstract

The trans-acyltransferase (AT) polyketide synthases are a recently recognised group of bacterial enzymes that generate complex polyketides. A prerequisite for re-engineering these poorly studied systems is knowledge about the substrate specificity of their components. In this work, KS domain 1 from the bacillaene polyketide synthase has been shown to possess high specificity towards 2-amidoacetyl intermediates, which are derived from incorporation of alpha amino acids into the polyketide chain. N-Acetylcysteamine (SNAC) analogues of full-length substrates were synthesised and incubated with the KS1 domain. The natural glycine-derived acyl–SNAC was found to acylate KS1 with highest efficiency, as evidenced by mass spectrometry (MS). An alanine variant was also incorporated, but its valine equivalent was not, which indicated limited tolerance of substitution at the α-position. Substrate analogues without an amine or amide nitrogen substituted on the 2-position were not accepted by KS1 at the standard assay concentration of 0.5 mM. Moreover, removal of Asn-206 from the active site of KS1 by site-directed mutagenesis reduced kcat/Km by a factor of approx. 2. This residue is conserved in most known 2-amidoacetyl-accepting KS domains from trans-AT PKSs and we postulate an important interaction between Asn-206 and the amide nitrogen of the substrate.