Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Engineering of Thermostable ?-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

ABSTRACT: The ?-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-?HAD), which catalyzes the NADP⁺ -dependent oxidation of ?-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192?L, N193?L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250?A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-?HAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50?°C.

SUBMITTER: Stockinger P

PROVIDER: S-EPMC7756219 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Engineering of Thermostable β-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

Stockinger Peter P Schelle Luca L Schober Benedikt B Buchholz Patrick C F PCF Pleiss Jürgen J Nestl Bettina M BM

Chembiochem : a European journal of chemical biology 20200916 24

The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-βHAD), which catalyzes the NADP<sup>+</sup> -dependent oxidation of β-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the sin ...[more]

PMID: 32939899

Similar Datasets

An (R)-Imine Reductase Biocatalyst for the Asymmetric Reduction of Cyclic Imines.

Project description:Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over-expressed the gene for an (R)-imine reductase [(R)-IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole-cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)-IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative-scale synthesis of the alkaloid (R)-coniine (90?% yield; 99?% ee) from the imine precursor was performed on a gram-scale. A homology model of the enzyme active site, based on the structure of a closely related (R)-IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis.

| S-EPMC4973613 | biostudies-literature

Asymmetric Reduction of (R)-Carvone through a Thermostable and Organic-Solvent-Tolerant Ene-Reductase.

Project description:Ene-reductases allow regio- and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene-reductase FOYE-1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE-1 to produce valuable compounds. A maximum activity of (319.7±3.2)?U?mg-1 with NADPH or of (206.7±3.4)?U?mg-1 with 1-benzyl-1,4-dihydronicotinamide (BNAH) for the reduction of N-methylmaleimide was observed at 30?°C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE-1 showed excellent performance in most systems with up to 20?vol% solvent and at temperatures up to 40?°C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200?mL biotransformation of 750?mg (R)-carvone afforded 495?mg of (2R,5R)-dihydrocarvone (>95?% ee), demonstrating the simplicity of handling and application of FOYE-1.

| S-EPMC7216909 | biostudies-literature

Catalytic asymmetric umpolung reactions of imines.

Project description:The carbon-nitrogen double bonds in imines are fundamentally important functional groups in organic chemistry. This is largely due to the fact that imines act as electrophiles towards carbon nucleophiles in reactions that form carbon-carbon bonds, thereby serving as one of the most widely used precursors for the formation of amines in both synthetic and biosynthetic settings. If the carbon atom of the imine could be rendered electron-rich, the imine could react as a nucleophile instead of as an electrophile. Such a reversal in the electronic characteristics of the imine functionality would facilitate the development of new chemical transformations that convert imines into amines via carbon-carbon bond-forming reactions with carbon electrophiles, thereby creating new opportunities for the efficient synthesis of amines. The development of asymmetric umpolung reactions of imines (in which the imines act as nucleophiles) remains uncharted territory, in spite of the far-reaching impact such reactions would have in organic synthesis. Here we report the discovery and development of new chiral phase-transfer catalysts that promote the highly efficient asymmetric umpolung reactions of imines with the carbon electrophile enals. These catalysts mediate the deprotonation of imines and direct the 2-azaallyl anions thus formed to react with enals in a highly chemoselective, regioselective, diastereoselective and enantioselective fashion. The reaction tolerates a broad range of imines and enals, and can be carried out in high yield with as little as 0.01 mole per cent catalyst with a moisture- and air-tolerant operational protocol. These umpolung reactions provide a conceptually new and practical approach to chiral amino compounds.

| S-EPMC4513368 | biostudies-literature

The D-2-hydroxyacid dehydrogenase incorrectly annotated PanE is the sole reduction system for branched-chain 2-keto acids in Lactococcus lactis.

Project description:Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V(max)/K(m) ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD(+) necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

| S-EPMC2632062 | biostudies-literature

Asymmetric allylboration of acyl imines catalyzed by chiral diols.

Project description:Chiral BINOL-derived diols catalyze the enantioselective asymmetric allylboration of acyl imines. The reaction requires 15 mol % (S)-3,3'-Ph2-BINOL as the catalyst and allyldiisopropoxyborane as the nucleophile. The reaction products are obtained in good yields (75-94%) and high enantiomeric ratios (95:5-99.5:0.5) for aromatic and aliphatic imines. High diastereoselectivities (diastereomeric ratio > 98:2) and enantioselectivities (enantiomeric ratio > 98:2) are obtained in the reactions of acyl imines with crotyldiisopropoxyboranes. This asymmetric transformation is directly applied to the synthesis of Maraviroc, the selective CCR5 antagonist with potent activity against HIV-1 infection. Mechanistic investigations of the allylboration reaction including IR, NMR, and mass spectrometry studies indicate that acyclic boronates are activated by chiral diols via exchange of one of the boronate alkoxy groups with activation of the acyl imine via hydrogen bonding.

| S-EPMC2638762 | biostudies-literature

Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly <b>Efficient</b> and Robust NADPH Regeneration System.

Project description:Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174-Pro178, located at the C-terminus of β7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxDHARRA for NADP (K cat/K M)NADP was 44.1 μM-1 min-1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxDHARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

| S-EPMC8047080 | biostudies-literature

Stereocontrol in asymmetric ?-lactam syntheses from imines and cyanosuccinic anhydrides.

Project description:Computations (SCS-MP2//B3LYP) reveal that the asymmetric synthesis of highly substituted ?-lactams with three stereogenic centers, including one quaternary center, proceeds through a Mannich reaction between the enol form of the anhydride and the E-imine, followed by a transannular acylation. This new mechanistic picture accounts for both the observed reactivity and stereoselectivity. CH-O and hydrogen bonding interactions in the Mannich step and torsional steering effects in the acylation step are responsible for stereocontrol. It is demonstrated that this new mechanistic picture applies to the related reactions of homophthalic anhydrides with imines and presents new vistas for the design of a new reaction to access complex molecular architectures.

| S-EPMC3988202 | biostudies-literature

Palladium-catalyzed asymmetric [3+2] cycloaddition of trimethylenemethane with imines.

Project description: Not available

| S-EPMC2532588 | biostudies-literature

Engineering an ultra-thermostable β(1)-adrenoceptor.

Project description:Conformational thermostabilisation of G-protein-coupled receptors is a successful strategy for their structure determination. The thermostable mutants tolerate short-chain detergents, such as octylglucoside and nonylglucoside, which are ideal for crystallography, and in addition, the receptors are preferentially in a single conformational state. The first thermostabilised receptor to have its structure determined was the β(1)-adrenoceptor mutant β(1)AR-m23 bound to the antagonist cyanopindolol, and recently, additional structures have been determined with agonist bound. Here, we describe further stabilisation of β(1)AR-m23 by the addition of three thermostabilising mutations (I129V, D322K, and Y343L) to make a mutant receptor that is 31 °C more thermostable than the wild-type receptor in dodecylmaltoside and is 13 °C more thermostable than β(1)AR-m23 in nonylglucoside. Although a number of thermostabilisation methods were tried, including rational design of disulfide bonds and engineered zinc bridges, the two most successful strategies to improve the thermostability of β(1)AR-m23 were an engineered salt bridge and leucine scanning mutagenesis. The three additional thermostabilising mutations did not significantly affect the pharmacological properties of β(1)AR-m23, but the new mutant receptor was significantly more stable in short-chain detergents such as heptylthioglucoside and denaturing detergents such as SDS.

| S-EPMC3819908 | biostudies-literature

Enantioselective reduction of sulfur-containing cyclic imines through biocatalysis.

Project description:The 3-thiazolidine ring represents an important structural motif in life sciences molecules. However, up to now reduction of 3-thiazolines as an attractive approach failed by means of nearly all chemical reduction technologies for imines. Thus, the development of an efficient general and enantioselective synthetic technology giving access to a range of such heterocycles remained a challenge. Here we present a method enabling the reduction of 3-thiazolines with high conversion and high to excellent enantioselectivity (at least 96% and up to 99% enantiomeric excess). This technology is based on the use of imine reductases as catalysts, has a broad substrate range, and is also applied successfully to other sulfur-containing heterocyclic imines such as 2H-1,4-benzothiazines. Moreover the effiency of this biocatalytic technology platform is demonstrated in an initial process development leading to 99% conversion and 99% enantiomeric excess at a substrate loading of 18 g/L in the presence of designer cells.

| S-EPMC5955971 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data