Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase

Background: Polyketides are compounds that possess medically significant ac tivities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs, Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mecha nisms that operate in natural PKS modules. Results: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative o f the erythromycin PKS that contains only modules 1 and 2. The reduction pr oducts of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S,3R) and (2R,3S) isomers of the corresponding P-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only con tains KR1 produced only the (2S,3R) isomer. Conclusions: In contrast with earlier results, KR1 selects only the (2S) is omer and reduces it stereospecifically to the (2S,3R)-3-hydroxy-2-methyl ac yl product, The KR domain of module 1 controls the stereochemical outcome a t both methyl- and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester ge nerated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereochemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, oper ate in modules 1 and 2 of the erythromycin PKS. These results should provid e a more rational basis for designing hybrid PKSs to generate altered stere ochemistry in polyketide products.