Imine Reductase Research Articles

Neutralizing the detrimental effect of glutathione on precious metal catalysts.

We report our efforts to enable transition-metal catalysis in the presence of cellular debris, notably Escherichia coli cell free extracts and cell lysates. This challenging goal is hampered by the presence of thiols, mainly present in the form of glutathione (GSH), which poison precious metal catalysts. To overcome this, we evaluated a selection of oxidizing agents and electrophiles toward their potential to neutralize the detrimental effect of GSH on a Ir-based transfer hydrogenation catalyst. While the bare catalyst was severely inhibited by cellular debris, embedding the organometallic moiety within a host protein led to promising results in the presence of some neutralizing agents. In view of its complementary to natural enzymes, the asymmetric imine reductase based on the incorporation of a biotinylated iridium pianostool complex within streptavidin (Sav) isoforms was selected as a model reaction. Compared to purified protein samples, we show that pretreatment of cell free extracts and cell lysates containing Sav mutants with diamide affords up to >100 TON's and only a modest erosion of enantioselectivity.

ChemInform Abstract: Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole‐Cell Biocatalyst Containing an (S)‐Imine Reductase.

ChemInform Abstract: Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole‐Cell Biocatalyst Containing an (S)‐Imine Reductase.

Computational Insights on an Artificial Imine Reductase Based on the Biotin–Streptavidin Technology

We present a computational study that combines protein–ligand docking, quantum mechanical, and quantum mechanical/molecular mechanical calculations to scrutinize the mechanistic behavior of the first artificial enzyme able to enantioselectively reduce cyclic imines. We applied a novel strategy that allows the characterization of transition state structures in the protein host and their associated reaction paths. Of the most striking results of our investigation is the identification of major conformational differences between the transition state geometries of the lowest energy paths leading to (R)- and (S)-reduction products. The molecular features of (R)- and (S)-transition states highlight distinctive patterns of hydrophobic and polar complementarities between the substrate and the binding site. These differences lead to an activation energy gap that stands in very good agreement with the experimentally determined enantioselectivity. This study sheds light on the mechanism by which transfer hydrogenases operate and illustrates how the change of environment (from homogeneous solution conditions to the asymmetric protein frame) affect the reactivity of the organometallic cofactor. It provides novel insights on the complexity in integrating unnatural organometallic compounds into biological scaffolds. The modeling strategy that we pursued, based on the generation of “pseudo transition state” structures, is computationally efficient and suitable for the discovery and optimization of artificial enzymes. Alternatively, this approach can be applied on systems for which a large conformational sampling is needed to identify relevant transition states.

An Artificial Imine Reductase based on the Ribonuclease S Scaffold

AbstractDative anchoring of a piano‐stool complex within ribonuclease S resulted in an artificial imine reductase. The catalytic performance was modulated upon variation of the coordinating amino acid residues in the S‐peptide. Binding of Cp*Ir (Cp*=C5Me5) to the native active site resulted in good conversions and moderate enantiomeric excess values for the synthesis of salsolidine.

Expanding the Chemical Diversity in Artificial Imine Reductases Based on the Biotin–Streptavidin Technology

AbstractWe report on the optimization of an artificial imine reductase based on the biotin‐streptavidin technology. With the aim of rapidly generating chemical diversity, a novel strategy for the formation and evaluation of biotinylated complexes is disclosed. Tethering the biotin‐anchor to the Cp* moiety leaves three free coordination sites on a d6 metal for the introduction of chemical diversity by coordination of a variety of ligands. To test the concept, 34 bidentate ligands were screened and a selection of the 6 best was tested in the presence of 21 streptavidin (Sav) isoforms for the asymmetric imine reduction by the resulting three legged piano stool complexes. Enantiopure α‐amino amides were identified as promising bidentate ligands: up to 63 % ee and 190 turnovers were obtained in the formation of 1‐phenyl‐1,2,3,4‐tetrahydroisoquinoline with [IrCp*biotin(L‐ThrNH2)Cl]⊂SavWT as a catalyst.

Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes

Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

Inside Cover: Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole-Cell Biocatalyst Containing an (S)-Imine Reductase (ChemCatChem 12/2013)

Inside Cover: Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole-Cell Biocatalyst Containing an (<i>S</i>)-Imine Reductase (ChemCatChem 12/2013)

High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.

Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole‐Cell Biocatalyst Containing an (S)‐Imine Reductase

Biocatalytic imine reduction: A whole-cell recombinant E. coli system, producing an (S)-selective imine reductase (IRED) from Streptomyces sp. GF3546, is developed. This biocatalyst is used for the enantioselective reduction of a broad range of substrates such as dihydroisoquinolines and dihydro-β-carbolines as well as iminium ions. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin–Streptavidin Technology

Artificial metalloenzymes enable the engineering of the reaction microenvironment of the active metal catalyst by modification of the surrounding host protein. We report herein the optimization of an artificial imine reductase (ATHase) based on biotin–streptavidin technology. By introduction of lipophilic amino acid residues around the active site, an 8-fold increase in catalytic efficiency compared with the wild type imine reductase was achieved. Whereas substrate inhibition was encountered for the free cofactor and wild type ATHase, two engineered systems exhibited classical Michaelis–Menten kinetics, even at substrate concentrations of 150 mM with measured rates up to 20 min–1.

Imine Reductases: A Comparison of Glutamate Dehydrogenase to Ketimine Reductases in the Brain

A key intermediate in the glutamate dehydrogenase (GDH)-catalyzed reaction is an imine. Mechanistically, therefore, GDH exhibits similarities to the ketimine reductases. In the current review, we briefly discuss (a) the metabolic importance of the GDH reaction in liver and brain, (b) the mechanistic similarities between GDH and the ketimine reductases, (c) the metabolic importance of the brain ketimine reductases, and (d) the neurochemical consequences of defective ketimine reductases. Our review contains many historical references to the early work on amino acid metabolism. This work tends to be overlooked nowadays, but is crucial for a contemporary understanding of the central importance of ketimines in nitrogen and intermediary metabolism. The ketimine reductases are important enzymes linking nitrogen flow among several key amino acids, yet have been little studied. The cerebral importance of the ketimine reductases is an area of biomedical research that deserves far more attention.

A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression

A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6nmol min(-1) mg(-1), and their optical purities were 92.7% ee, 96.4% ee, and >99% ee, respectively. Using a NADPH-regenerating system, 10mM 2-MPN was converted to amine with 100% conversion and 92% ee after 24h. The amino acid sequence analysis revealed that SIR showed about 60% identity to 6-phosphogluconate dehydrogenase. However, it showed only 37% identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.

Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo (1.85 Å) and NADP(+)-bound (2.31 Å) forms. Irp3 is structurally homologous to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-d-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologues that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism for the donation of a proton from a general acid (either histidine 101 or tyrosine 128) and the donation of a hydride from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline.

Purification and Characterization of a Novel (R)-Imine Reductase fromStreptomycessp. GF3587

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.

Artificial Transfer Hydrogenases for the Enantioselective Reduction of Cyclic Imines

Man-made activity: Introduction of a biotinylated iridium piano stool complex within streptavidin affords an artificial imine reductase (see scheme). Saturation mutagenesis allowed optimization of the activity and the enantioselectivity of this metalloenzyme, and its X-ray structure suggests that a nearby lysine residue acts as a proton source during the transfer hydrogenation.

Biotransformations

This report reviews significant developments in applications of biological catalysis in synthetic organic chemistry for the year 2010. Hydrolases have been applied in kinetic resolution and quantitative DKR processes and have been evolved in vitro to give enzymes displaying improved enantioselectivity and solvent stability. Applications of reductases have included an unusual instance of the generation of optically active atropisomers and also the identification of stereoselective imine reductases in Streptomyces spp. Other biotransformations leading to optically active amines have focused on transaminases, and a wide-ranging genomics analysis has revealed nineteen new and previously rare (R)-selective transaminases. A further (R)-selective transaminase was extensively engineered for process suitability for application in the industrial synthesis of sitagliptin. Improved carbon–carbon bond formation by aldolases is being addressed through the identification and improvement of enzymes accepting non-phosphorylated dihydroxyacetone as a substrate donor for the synthesis of sugars. In addition, new enzymatic activities, including natural enzymes that catalyse the Stetter reaction, and also the Michael-type addition of water to cyclohexenone have been reported. Finally, advances in the in silico design of enzymes are proceeding rapidly, with the report of the design of the first enzyme known to catalyse an bimolecular Diels-Alder reaction.

Asymmetric synthesis of chiral cyclic amine from cyclic imine by bacterial whole-cell catalyst of enantioselective imine reductase

Streptomyces sp. GF3587 and 3546 were found to be imine-reducing strains with high R- and S-selectivity by screening using 2-methyl-1-pyrroline (2-MPN). Their whole-cell catalysts produced 91 mM R-2-methylpyrrolidine (R-2-MP) with 99.2%e.e. and 27.5 mM S-2-MP (92.3%e.e.) from 2-MPN at 91-92% conversion in the presence of glucose, respectively.

A dynamic combinatorial screen for novel imine reductase activity

New imine reductase activity has been discovered in the anaerobic bacterium Acetobacterium woodii by screening a dynamic combinatorial library of virtual imine substrates, using a biphasic water–tetradecane solvent system. Benzylidine aniline and butylidine aniline were reduced to the corresponding amines by caffeate-induced cells, whereas uninduced cells reduced butylidine aniline only. The reductions were detected despite side reactions that consumed some of the starting materials. The new screen can now be extended to discover synthetically useful imine reductases and enzymes that catalyse reactions for which biocatalytic equivalents of the chemical reactions have not yet been discovered.