Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates

Background: Polyketides are structurally diverse natural products with a ra nge of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) in which eac h cycle of chain extension requires a different 'module' of enzymatic activ ities. Attempts to design and construct modular PKSs that synthesise specif ied novel polyketides provide a particularly stringent test of our understa nding of PKS structure and function. Results: We show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an une xpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construc t a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketos ynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprisin g. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisi ngly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively. Conclusions: These results demonstrate that the epimerising activity associ ated with module 1 of the erythromycin PKS can be conferred on module 5 mer ely by transfer of the KS1 domain. Moreover, the normally precise stereoche mical control observed in modular PKSs is lost when KR5 and KR6 are challen ged by an unfamiliar substrate, which is much smaller than their natural su bstrates. This observation demonstrates that the stereochemistry of ketored uction is not necessarily invariant for a given KR domain and underlines th e need for mechanistic understanding in designing genetically engineered PK Ss to produce novel products. (C) 2001 Elsevier Science Ltd. All rights res erved.