Structural and Mutagenesis Studies of the Thiamine‐Dependent, Ketone‐Accepting YerE from Pseudomonas protegens

Abstract

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.

Unifying Scheme for the Biosynthesis of Acyl‐Branched Sugars: Extended Substrate Scope of Thiamine‐Dependent Enzymes

“…Both the decrease in the ee value of 25 and the formation of the minor diastereomer of 26 might result from a nonenzymatic racemization of the benzylic stereocenter, as is well-known for 1-hydroxy-1-arylpropan-2-ones. [15] According to our proposed unifying scheme, the biosynthetic strategy should also be applicable to the other aforementioned longer-branched sugars such as caryophyllose (10) or gastriose (13). Alibaud et al knocked out several genes of the putative BGC of caryophyllose, while studying the biosynthesis of LOS from M. marinum.…”

Recent Developments in the Synthesis of β-Diketones

“…An enzyme homologous (59% amino acid identity) to YpYerE, PpYerE from Pseudomonas protegens has been recently identified, crystallized, and its 3D structure has been determined [116]. Anyhow, comparing yields and ee values reported in the preparation of compounds 94b and 96, results with PpYerE are not better than those obtained with YpYerE.…”

Building Up Quaternary Stereocenters Through Biocatalyzed Direct Insertion of Carbon Nucleophiles on Ketones

“…Moreover, thanks to the determination of the protein sequence, an enzyme homologous (59 % amino acid identity) to Yp YerE, Pp YerE from Pseudomonas protegens with similar catalytic activity has been identified, crystallized and its structure has been determined. The potential binding pocket for the acceptor substrate, close to the active site, also referred as the “S‐pocket” common to many ThDP‐dependent decarboxylases, has been identified and modified by a few amino acids exchange.…”

“…A comparison of Pp YerE with Yp YerE as regard the acceptor ketones scope, with pyruvate as the donor, is reported in Table …”