Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase.

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated ß-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.

Addition of a polyhistidine tag alters the regioselectivity of carbonyl reductase S1 from Candida magnoliae.

Studying enzymatic reductions of substrates with more than a single keto group is challenging, as the carbonyl reduction can create a vast array of regio- and stereoisomers. If used as reference compounds, regio- and stereopure hydroxy ketides could facilitate the characterization of reductases with unclear regio- and stereoselectivity. We have combined nonenzymatic and enzymatic reduction and oxidation steps to obtain all four regio- and stereoisomers of tert-butyl hydroxyoxohexanoates in high optical purity (enantiomeric ratio (er) of 99 : 1 for the δ-hydroxy-ß-keto isomers; er of >97 : 3 for the ß-hydroxy-δ-keto isomers). Furthermore, we have prepared seven of the eight possible regioisomers and diastereomers of γ-methylated hydroxyoxohexanoates. These 11 compounds allowed unraveling the complex stereoselectivity of ß,δ-diketo ester reductions catalyzed by carbonyl reductase S1 from Candida magnoliae (CMCR-S1). Our analysis shows that the regio- and stereoselectivity of CMCR-S1-catalyzed reductions is highly sensitive toward modifications at the C-terminus of CMCR-S1: in addition to the expected δ-hydroxy product, the variant with a C-terminal His-tag also led to formation of ß-hydroxy by-products with high optical purity.

Regio- and stereoselective intermolecular oxidative phenol coupling in Streptomyces.

Intermolecular oxidative phenol coupling is the main process in nature for the formation of atroposelective biaryl compounds. Although well defined in plants and fungi, this type of dimerization reaction in bacteria is poorly understood. Therefore, the biosynthesis of julichromes, spectomycins, and setomimycin was investigated. The monomeric subunits of these biarylic pre-anthraquinones are derived from a common polyketidic precursor, yet the coupling reaction proceeds in a regioselective manner, with the position of attachment of the two subunits depending on the specific streptomycete strain. By using genome analysis and deletion experiments, the biosynthetic gene clusters were identified. Furthermore, it was established that cytochrome P450 enzymes are fundamentally involved during dimerization of the polyketide monomers.

Substrate-dependent stereospecificity of Tyl-KR1: an isolated polyketide synthase ketoreductase domain from Streptomyces fradiae.

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-ß-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.