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The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.

Thiamine diphosphate (ThDP)-dependent enzymes are wellestablished catalysts in the field of asymmetric synthesis. [1] One of the model reactions catalyzed by these enzymes is the asymmetric CÀC bond-formation reaction between two aldehyde substrates that leads to the formation of 2-hydroxyketones in high enantioselectivities. [2] Exchange of one of the aldehyde substrates in this reaction for a ketone [3] would offer the opportunity for the catalytic asymmetric formation of chiral tertiary alcohols, which are important structural units in natural products and bioactive agents. [4,5] During the last decade, different organocatalysts have been developed for the asymmetric aldehyde-ketone cross-coupling reaction, [6] and intramolecular variants of this reaction have been reported in the literature. [7,8] Most recently, Enders and Henseler described the direct intermolecular cross-coupling between aldehydes and trifluoromethyl ketones using a bicyclic triazolium salt as the catalyst. [9] The asymmetric intermolecular non-enzymatic coupling reaction with ketone acceptors should be more difficult, owing to the lower electrophilicity of the ketone carbonyl group, and the increased steric hindrance compared with aldehyde substrates, and has not yet been reported in the literature. Herein, we present an asymmetric intermolecular aldehyde-ketone carboligation reaction using a ThDP-dependent enzyme as the catalyst (Scheme 1).Branched-chain sugars are important bioactive carbohydrates and are widely represented in nature. Using feeding experiments, it can be shown that the two-carbon branch of several of these sugar derivatives is derived from pyruvate. In 1972, Grisebach and Schmid postulated the participation of ThDP in this carboligation reaction. [10] In the biosynthetic pathway of yersiniose A, a two-carbon branched-chain 3,6di(deoxy)hexose that is found in the O-antigen of Yersinia pseudotuberculosis O:VI, the ThDP-dependent flavoenzyme YerE catalyzes the decarboxylation of pyruvate and the transfer of the activated acetaldehyde onto the carbonyl function of CDP-3,6-di(deoxy)-4-keto-d-glucose (CDP = cytidine-5'-diphosphate). [11,12] The enzymatic activity of YerE was confirmed by incubation of the protein with the enzymatically prepared physiological substrate (starting from CDP-d-glucose). The isolated product CDP-4-aceto-3,6dideoxygalactose was confirmed by NMR spectroscopy.To analyze the substrate range of the enzyme, we amplified the gene yerE from the chromosomal DNA of Y. pseudotuberculosis O:VI using a polymerase chain reaction. The gene was cloned into the pQE-60 expression vector and the recombinant protein was produced in Escherichia coli BL21(DE3) cells. The overexpressed C-terminal His-tagged YerE protein was purified to homogeneity by affinity chromatography using an Ni-NTA purification system (see the Supporting Information).The amino acid sequence of YerE is similar to that of the large subunit of E. coli acetohydroxyacid synthases (EcAHAS) (31 % identity). AHAS successfully catalyzed the ThDP-dependent ...

A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 Å.

Thiamine diphosphate-dependent enzymes catalyze the formation of C-C bonds, thereby generating chiral secondary or tertiary alcohols. By the use of vibrational circular dichroism (VCD) spectroscopy we studied the stereoselectivity of carboligations catalyzed by YerE, a carbohydrate-modifying enzyme from Yersinia pseudotuberculosis. Conversion of the non-physiological substrate (R)-3-methylcyclohexanone led to an R,R-configured tertiary alcohol (diastereomeric ratio (dr) >99:1), whereas the corresponding reaction with the S enantiomer gave the S,S-configured product (dr>99:1). This suggests that YerE-catalyzed carboligations can undergo either an R- or an S-specific pathway. We show that, in this case, the high stereoselectivity of the YerE-catalyzed reaction depends on the substrate's preference to acquire a low-energy conformation.