Predicting the nature and timing of epimerisation on a modular polyketide synthase.

The modular polyketide synthase leading to the formation of the erythromycin aglycone 6-deoxyerythronolide B (6dEB) in Saccharopolyspora erythraea is one of the best studied, especially regarding stereochemistry. Biosynthesis proceeds by Claisen condensation with inversion of configuration to give a 2R methyl centre. However, the methyl stereochemistry of 6dEB implies that two epimerisation steps occur. We have taken a bioinformatics approach to explore the mechanisms for this bifunctionality. 2] Polyketides are assembled through serial condensations of activated coenzyme A thioester monomers derived from simple organic acids such as acetate, propionate and butyrate. The activities required for each cycle of condensation and processing of the b-keto group are contained within a module, and a discrete domain represents each activity. The active sites required for condensation include an acyltransferase (AT), an acyl carrier protein (ACP) and a b-ketoacylsynthase (KS). Each condensation results in a b-keto group that undergoes all, some or none of a series of processing steps. Active sites that perform these reactions are contained within the ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains. The absence of any b-keto processing results in the incorporation of a ketone group into the growing polyketide chain, a KR alone gives rise to a hydroxyl moiety, a KR and a DH produce an alkene, while the combination of KR, DH and ER domains lead to complete reduction to an alkane. After assembly, the polyketide chain typically undergoes cyclisation and post-PKS modifications to give the final active compound. The enzymic steps leading to the formation of 6-dEB are ACHTUNGTRENNUNGencoded within six distinct modules. These modules, together with a loading domain and a chain-terminating thioesterase (TE) domain are housed within three polypeptides that collectively catalyse the condensation and appropriate reduction of one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. Modules 1, 2, 5 and 6 contain a KR domain; module 4 contains a complete set of reductive domains— namely KR, DH and ER; while module 3 has no reductive functionality. Polyketide biosynthesis proceeds by a Claisen condensation with inversion of configuration to give a 2R methyl centre. However, in module 1, this decarboxylative condensation involves cleavage of the CH bond adjacent to the methyl group at C2 of the extender unit to produce a 2S methyl centre. This implies that there must be an epimerisation step taking place, which would account for the combinations of methyl stereochemistry present in 6-dEB, namely 2R, 4R, 6S, 8R, 10R, 12S. It is possible that proton exchange between the bketoester and water could promote epimerisation, with downstream domains then displaying absolute specificity for only a given isomer; but how this reaction proceeds remains an enigma, since discrete epimerisation domains have never been discovered in these modular systems. It would be a very attractive proposition to discover cryptic epimerisation activities housed within already described domains. Two types of KR domains, termed A and B types, have been described. Recently, the crystal structure of the KR domain from module 1 of 6-dEB synthase (DEBS) was reported at 1.79 @ resolution. This protein folds to adopt two domains, one serving a purely structural role by stabilising the other domain, which catalyses reduction of the C3 carbonyl of the diketide. Both contain identical active-site regions buried within the protein core, but differ according to the presence of a D residue on one side of the active-site groove that is thought to direct the substrate thioester carbonyl towards the amino group of a conserved Y, catalysing an S reduction by the B forms. In the A forms, this D residue is absent or replaced by W so that the direction of hydride addition is to the Si rather than Re face of the keto group, thus giving rise to an R chirality. The authors also suggest that a Y residue, conserved in all KR domains, is flanked by amino acids in epimerising modules allowing flexibility to catalyse epimerisation of the methyl stereocentre at C2. This Y can act as a base, extracting a proton from the diketide substrate to form an enolate. Proton addition to the enolate oxygen releases the diketide from the KR, but this epimerised diketide can undergo a tautomeric shift back to the keto form, presumably acting as an additional substrate for the KR. We believe that this mechanism would stall further polyketide chain synthesis, which seems unlikely given that the overall Km/Kcat for 6-dEB synthesis is relatively rapid for a multifunctional enzyme. Here we take a bioinformatics approach to explore an alternative mechanism that KR domains contain a bifunctional active site that can fix both the methyl and alcohol stereochemistry of b-ketoacyl-ACP intermediates (Figure 1). Family profiling was first undertaken to predict the most likely location for epimerisation within the domains of the DEBS proteins. Sequence comparisons were then used to identify particular amino acids that could be involved in this catalysis. Family profiling within DEBS confirmed the sequence positions of all the known catalytic domains. Interestingly, however, [a] Dr. P. F. Long The School of Pharmacy, University of London 29/39 Brunswick Square, London WC1N 1AX (UK) Fax: (+44)207-753-5868 E-mail : [email protected] [b] A. Starcevic, Prof. Dr. D. Hranueli Section for Bioinformatics, Department of Biochemical Engineering Faculty of Food Technology and Biotechnology, University of Zagreb Pierottijeva 6, 10000 Zagreb (Croatia) [c] Prof. M. Jaspars Department of Chemistry, University of Aberdeen Old Aberdeen AB24 3UE (UK) [d] Prof. Dr. J. Cullum Department of Genetics, University of Kaiserslautern Postfach 3049, 67653 Kaiserslautern (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.