Exploring donor substrate promiscuity of a Thermostable Transketolase by directed evolution

Enzymes catalyzing asymmetric carboligation reactions typically show very high specificity for their nucleophilic substrate. Transketolase (TK, EC 2.2.1.1) catalyses a reversible transfer of a hydroxylated C2 fragment among phosphorylated ketoses and aldoses. [1] Native TK converts a large variety of (2R)-hydroxyaldehydes as the electrophilic acceptor substrates, but apart from its natural phosphoketose donors TK accepts only hydroxy­pyruvate (hydroxylated donor) (Figure 1). In contrast, 1-deoxy-D-xylulose-5-phosphate synthase (DXS, EC 2.2.1.7) catalyzes the specific decarboxylative transfer of the acetyl moiety from pyruvate (non-hydroxylated donor) to glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose 5-phosphate (DXP), which constitutes the first step into the non-mevalonate biosynthesis of terpenoids (Figure 1).[2] Reactions of native TK and DXS are mutually exclusive in vivo. Please click Additional Files below to see the full abstract

Evidence that waste aversion begets insurance aversion

Paying an insurance premium but not needing to claim is sometimes viewed as pouring money down the drain. Aversion to the perceived waste may lead to the rejection of fair insurance. Although policies paying rebates if no claim is made are not attractive to expected utility maximisers, this paper finds strong evidence they appeal to waste averters.

Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k-cat/K-m toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics

Hydroxamate Assays for High‐Throughput Screening of Transketolase Libraries Against Arylated Substrates

We recently reported that the transketolase from Geobacillus stearothermophilus (TKgst) upon acyl transfer to nitrosoarenes generates N‐aryl hydroxamic acids (HA). The latter are metal chelating compounds that in the presence of Fe(III) ions form deep‐red complexes. Here, we applied this principle to the development of a colorimetric assay in both solid‐ and liquid‐phase formats for the high‐throughput screening of TKgst and its variants. Screening a set of positive hits from a L382X/D470X library validated the specificity and sensitivity of the assays. The solid surface assay allows a clear distinction between positive and negative colonies by the naked eye in qualitative mode, and further also to measure activity in semi‐quantitative fashion in the liquid‐phase format. The assay will be important for engineering the TKgst enzyme towards improved conversion of aromatic aldehydes by their close structural analogy to nitrosoarenes

Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva.

Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. In the present work, we identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, we reconstructed the main pathway that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. Our results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation

Combining aldolases and transaminases for the synthesis of 2‑amino-4-hydroxybutanoic acid

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee values of >99%.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.), and COST action CM1303 Systems Biocatalysis.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe