The avermectin-producing polyketide synthase

• Main Author: Galloway, I. S.
• Published: University of Cambridge 1998
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599283

In this work, sequencing of an approximately 14kb region of the avermectin cluster has revealed that the first two modules are apparently housed in one polypeptide, AVES1. Comparison of this sequence with those from other PKSs has indicated a region of the acyl transferase (AT) domain that can be used to predict whether the domain's polyketide chain-extender unit specificity is for malonyl CoA or methyl-malonyl CoA. Further analysis showed an exact correlation between all the other domains required for completion of the first two polyketide chain-extension cycles, as predicted from the structure of avermectin and those found in AVES1, except that the dehydratase domain (DH) in module 2 appeared to lack part of the active site motif. Dehydration at this point in avermectins (C-22,23) is associated with anthelminthic activity. To probe the function of the module 2 DH the avermectin PKS was truncated by relocating the chain-releasing thioesterase (TE), from the C-terminal end of the final module in the erythromycin-producing PKS, to the C-terminal end of AVES1. The resultant hybrid PKS (AVES1TE), when expressed heterologously in a host containing no PKS genes, appeared to exclusively produce 3-hydroxy-δ-lactones. This provides strong evidence for the notion that the partial C-22,23-dehydration of avermectins normally observed in <I>Streptomyces avermitilis </I>is not due to the AVES DH2 and is consistent with recent reports that the dehydration may be catalysed by the <I>aveC</I> gene product. The functions of individual domains, including β-keto acyl synthase domains (KS), acyl carrier protein domains (ACP) and AT domains, from the avermectin PKS were investigated by substituting them for analogous domains in a previously-established and truncated version of the erythromycin-producing PKS (DEBSITE), upon which AVES1TE was modelled. These experiments have shed light on the controlling factors that determine the stereochemistry of the extending chain.