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

Abstract

These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.

A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

“…For example, the reductive loop of DEBS module 5, which in its native context gives a (2R,3S) configuration in the alcohol product, was cloned into AvrII-HpaI sites (in S. erythraea JC2, pJCE5), and the fermentation of the resulting strain gave comparable yield and identical products to the control derived from DEBS module 2. This outcome makes a telling contrast to the results of previous in vivo work, [30] in which KS 5 was replaced by the DEBS loading module and KS 1 in DEBS 3. The products of that hybrid multienzyme included not only the normal products of DEBS3, but also an aberrant triketide lactone.…”

“…[30] In the present case, bringing KR 5 into a chimaeric module with KS 2 did not lead to any loss of stereospecificity or stereoselectivity; this supports a model for catalysis [26,27,30] in which DEBS KS 2 differs from KS 1 in that it cannot catalyse the epimerisation of the (2R)-2-methyl-3-ketopentanoyl-ACP intermediate, or cannot do so fast enough to compete with ketoreduction. This experiment also shows that the ACP-tethered diketide is reduced with full fidelity by KR 5 , even though previous in vitro studies have shown that recombinant purified KR 5 reduces the (untethered) racemic substrate (2RS)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester to give three stereoisomeric products.…”

“…For example, this can occur if a KR within a chimaeric extension module is presented only with a single stereoisomer of the 2-methyl-3-ketoacyl thioester intermediate against which it is inactive. [30] Conversely the KR in its new context may be given access to both 2-methyl stereoisomers, which allows the intrinsic KR selectivity to dictate the outcome at both C-2 and C-3 stereocentres. [30] We present here the results of introducing synthetic oligonucleotide polylinkers into the DNA encoding DEBS 1-TE in place of the KR 2 domain, and their use in splicing heterologous "reductive loops" to obtain hybrid PKSs that synthesise triketide lactones differing in oxidation level or (where the C-3 hydroxyl is retained) in C-3 alcohol and C-2 methyl stereochemistry.…”

“…[30] Conversely the KR in its new context may be given access to both 2-methyl stereoisomers, which allows the intrinsic KR selectivity to dictate the outcome at both C-2 and C-3 stereocentres. [30] We present here the results of introducing synthetic oligonucleotide polylinkers into the DNA encoding DEBS 1-TE in place of the KR 2 domain, and their use in splicing heterologous "reductive loops" to obtain hybrid PKSs that synthesise triketide lactones differing in oxidation level or (where the C-3 hydroxyl is retained) in C-3 alcohol and C-2 methyl stereochemistry. Our findings shed further light on the substrate specificity and stereochemical course of catalysis in module 2 of DEBS and in the other extension modules whose reductive loops have been assayed.…”

Evaluating Ketoreductase Exchanges as a Means of Rationally Altering Polyketide Stereochemistry

“…Althoughi tw as initially proposed that the KS domains were also responsible for epimerization thus yieldingt he l-configuration observed in many polyketides, [13,14] it is now clear that lmethyl groups arise from the epimerase activity of certain KR domains, even when the KRs are inactive as ketoreductases. [15][16][17][18] This understanding has led to the classification of modular PKS KR domains into six types: [15] those catalyzing ketoreduction but not epimerization (A1 and B1), ketoreductiona nd epimerization (A2 and B2), epimerization but not reduction (C2), and fully inactive KRs (C1).…”