Substrate‐Dependent Stereospecificity of Tyl‐KR1: An Isolated Polyketide Synthase Ketoreductase Domain from Streptomyces fradiae

Abstract

The stereospecificity of an enzymatic reaction depends on the way in which a substrate and its enantiomer bind to the active site. These binding modes cannot be easily predicted. We have studied the stereospecificity and stereoselectivity of the ketoreductase domain Tyl-KR1 of the tylactone polyketide synthase from Streptomyces fradiae by analysing the stereochemical outcome of the reduction of five different keto ester substrates. The absolute configuration of the Tyl-KR1 reduction products was determined by using vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations. The conversion of only one of the tested substrates, 2-methyl-3-oxovaleric acid N-acetylcysteamine thioester, afforded the expected anti-(2R,3R) configuration of the α-methyl-β-hydroxyl ester product, representing the stereochemistry observed for the physiological polyketide product tylactone. For all other substrates, which were modified with respect to the type of ester and/or the chain length (C4 instead of C5), the opposite configuration (anti-(2S,3S)) was obtained with significant enantio- and diastereoselectivity. Inversion of both stereocentres suggests completely different binding modes invoked by only minor modifications of the substrate structure.

Exploiting Enzymatic Dynamic Reductive Kinetic Resolution (DYRKR) in Stereocontrolled Synthesis

“…One such example is reported by Häckh in the reduction of 2-Me-3-oxo esters (Scheme 3). [28] Replacement of an SNAC ester with an ethyl ester increased enantioselectivity from 43% ee to 92% ee. Interestingly, the same ester substitution for 2-Me-3-oxovalerate esters reversed the stereochemical course of the reduction from (2 R ,3 R ) to (2 S ,3 S ).…”

Substrate structure–activity relationships guide rational engineering of modular polyketide synthase ketoreductases

“…7 In contrast, studies in which the 1 st KR from the tylosin PKS (TylKR1) was incubated with 2-methyl-3-oxopentanoyl diketides linked to handles through an oxo-ester showed that reversals in stereocontrol can result from unnatural handles. 10 To further dissect the interactions that mediate KR stereocontrol, we varied the chemistry and size of the handles of 2-methyl-3-oxopentanoyl substrates and evaluated the effects in structure-activity relationship (SAR) fashion.…”

Structural and functional studies of a trans -acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α- and β-ketoreduction

“…To appreciate the extent to which PksKR1 is specialized in performing its role as a stereospecific α‐KR, we incubated the α‐keto substrate 2 with the biocatalytically robust cis ‐AT KR enzymes TylKR1 and EryKR1 [Fig. (b)] . EryKR1 was able to perform the stereospecific reduction of 2 to yield a product that has a mass consistent with the D‐α‐hydroxy reduction product.…”