498-18-0Relevant academic research and scientific papers
Rational engineering ofAcinetobacter tandoiiglutamate dehydrogenase for asymmetric synthesis ofl-homoalanine through biocatalytic cascades
Diao, Shiqing,Jiang, Shuiqin,Liu, Yan,Sun, Yangyang,Wang, Hualei,Wang, Liuzhu,Wei, Dongzhi
, p. 4208 - 4215 (2021/06/30)
l-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase fromAcinetobacter tandoii(AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity ofAtGluDH towards α-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 Ml-threonine, which is close to the solubility limit, we obtained a 99.9% yield ofl-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L?1h?1space-time yield. Additionally, the activities of the engineeredAtGluDH towards some other hydrophobic amino acids were also improved to 1.1-11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
Biocatalysed synthesis of chiral amines: continuous colorimetric assays for mining amine-transaminases
Gourbeyre, Léa,Heuson, Egon,Charmantray, Franck,Hélaine, Virgil,Debard, Adrien,Petit, Jean-Louis,de Berardinis, Véronique,Gefflaut, Thierry
, p. 904 - 911 (2021/02/26)
In the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral amines, we have developed new continuous assays for the screening of amine-transaminase collections. These assays are based on the use of hypotaurine as an irreversible amine donor. This β-aminosulfinic acid is converted upon transamination into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily detected by spectrophotometry using Ellman's reagent. Two complementary assays were developed based on this titration method. Firstly, a direct assay allowed detection of various transaminases able to use hypotaurine as an amino donor. In a second coupled assay,l-alanine is used as a generic donor substrate of amine-transaminases and is regenerated using an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a variety of valuable new biocatalysts for use in synthetic processes.
Bioelectrocatalytic Conversion from N2 to Chiral Amino Acids in a H2/α-Keto Acid Enzymatic Fuel Cell
Cai, Rong,Chen, Hsiaonung,Chen, Hui,Dong, Fangyuan,Minteer, Shelley D.,Prater, Matthew B.
supporting information, p. 4028 - 4036 (2020/03/11)
Enzymatic electrosynthesis is a promising approach to produce useful chemicals with the requirement of external electrical energy input. Enzymatic fuel cells (EFCs) are devices to convert chemical energy to electrical energy via the oxidation of fuel at the anode and usually the reduction of oxygen or peroxide at the cathode. The integration of enzymatic electrosynthesis with EFC architectures can simultaneously result in self-powered enzymatic electrosynthesis with more valuable usage of electrons to produce high-value-added chemicals. In this study, a H2/α-keto acid EFC was developed for the conversion from chemically inert nitrogen gas to chiral amino acids, powered by H2 oxidation. A highly efficient cathodic reaction cascade was first designed and constructed. Powered by an applied voltage, the cathode supplied enough reducing equivalents to support the NH3 production and NADH recycling catalyzed by nitrogenase and diaphorase. The produced NH3 and NADH were reacted in situ with leucine dehydrogenase (LeuDH) to generate l-norleucine with 2-ketohexanoic acid as the NH3 acceptor. A 92% NH3 conversion ratio and 87.1% Faradaic efficiency were achieved. On this basis, a H2-powered fuel cell with hyper-thermostable hydrogenase (SHI) as the anodic catalyst was combined with the cathodic reaction cascade to form the H2/α-keto acid EFC. After 10 h of reaction, the concentration of l-norleucine achieved 0.36 mM with >99% enantiomeric excess and 82% Faradaic efficiency. From the broad substrate scope and the high enzymatic enantioselectivity of LeuDH, the H2/α-keto acid EFC is an energy-efficient alternative to electrochemically produce chiral amino acids for biotechnology applications.
Semi-rational hinge engineering: modulating the conformational transformation of glutamate dehydrogenase for enhanced reductive amination activity towards non-natural substrates
Liu, Yayun,Meng, Lijun,Wu, Jianping,Yang, Lirong,Yin, Xinjian,Zhou, Haisheng
, p. 3376 - 3386 (2020/06/09)
The active site is the common hotspot for rational and semi-rational enzyme activity engineering. However, the active site represents only a small portion of the whole enzyme. Identifying more hotspots other than the active site for enzyme activity engineering should aid in the development of biocatalysts with better catalytic performance. Glutamate dehydrogenases (GluDHs) are promising and environmentally benign biocatalysts for the synthesis of valuable chirall-amino acids by asymmetric reductive amination of α-keto acids. GluDHs contain an inter-domain hinge structure that facilitates dynamic reorientations of the domains relative to each other. Such hinge-bending conformational motions of GluDHs play an important role in regulating the catalytic activity. Thus, the hinge region represents a potential hotspot for catalytic activity engineering for GluDHs. Herein, we report semi-rational activity engineering of GluDHs with the hinge region as the hotspot. Mutants exhibiting significantly improved catalytic activity toward several non-natural substrates were identified and the highest activity increase reached 104-fold. Molecular dynamics simulations revealed that enhanced catalytic activity may arise from improving the open/closed conformational transformation efficiency of the protein with hinge engineering. In the batch production of three valuablel-amino acids, the mutants exhibited significantly improved catalytic efficiency, highlighting their industrial potential. Moreover, the catalytic activity of several active site tailored GluDHs was also increased by hinge engineering, indicating that hinge and active site engineering are compatible. The results show that the hinge region is a promising hotspot for activity engineering of GluDHs and provides a potent alternative for developing high-performance biocatalysts toward chirall-amino acid production.
Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
Kim, Hong-Gon,Han, Sang-Woo,Shin, Jong-Shik
, p. 2594 - 2606 (2019/05/15)
ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., R-analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R-analysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2×104-fold activity increase for 1-phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with >99% ee of a resulting (S)-amine. (Figure presented.).
Stereoselective Synthesis of syn -γ-Hydroxynorvaline and Related α-Amino Acids
Berke?, Du?an,Caletková, O?ga,Ferko, Branislav,Jakubec, Pavol,Kolarovi?, Andrej,Puch?ová, Eva,Valachová, Dominika
, p. 4568 - 4575 (2019/12/11)
The total syntheses of three enantiomerically pure non-proteinogenic amino acids, l -norvaline, γ-oxonorvaline, and syn -γ-hydroxynorvaline, are reported. The chromatography-free route pivoted on the construction of highly enantiomerically enriched substituted α-amino-γ-oxopentanoic acid, from which all three members were accessed divergently via chemoselective and stereoselective reductions. The rapid synthesis of this key α-amino-γ-oxopentanoic acid was achieved by a highly diastereoselective crystallisation-driven three-component Mannich reaction from the readily available building blocks acetone, glyoxylic acid monohydrate, and (S)-(4-methoxyphenyl)ethylamine. The enantiomeric purity of all target molecules was confirmed by HPLC analysis, either of the amino acids or their derivatives.
Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids
Zhou, Haisheng,Meng, Lijun,Yin, Xinjian,Liu, Yayun,Xu, Gang,Wu, Jianping,Wu, Mianbin,Yang, Lirong
supporting information, p. 6470 - 6477 (2019/11/02)
Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri-enzyme one-pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l- and four d- unnatural amino acid products, using ammonia and the co-enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2-propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.
Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade
Zhang, Danping,Jing, Xiaoran,Zhang, Wenli,Nie, Yao,Xu, Yan
, p. 29927 - 29935 (2019/10/01)
d-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However, establishing a universal enzymatic method for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we constructed and optimized a cascade enzymatic route involving l-amino acid deaminase and d-amino acid dehydrogenase for the biocatalytic stereoinversions of l-amino acids into d-amino acids. Using l-phenylalanine (l-Phe) as a model substrate, this artificial biocatalytic cascade stereoinversion route first deaminates l-Phe to phenylpyruvic acid (PPA) through catalysis involving recombinant Escherichia coli cells that express l-amino acid deaminase from Proteus mirabilis (PmLAAD), followed by stereoselective reductive amination with recombinant meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) to produce d-phenylalanine (d-Phe). By incorporating a formate dehydrogenase-based NADPH-recycling system, d-Phe was obtained in quantitative yield with an enantiomeric excess greater than 99%. In addition, the cascade reaction system was also used to stereoinvert a variety of aromatic and aliphatic l-amino acids to the corresponding d-amino acids by combining the PmLAAD whole-cell biocatalyst with the StDAPDH variant. Hence, this method represents a concise and efficient route for the asymmetric synthesis of d-amino acids from the corresponding l-amino acids.
One-Pot Preparation of d-Amino Acids Through Biocatalytic Deracemization Using Alanine Dehydrogenase and Ω-Transaminase
Han, Sang-Woo,Shin, Jong-Shik
, p. 3678 - 3684 (2018/10/20)
d-Amino acids are pharmaceutically important building blocks, leading to a great deal of research efforts to develop cost-effective synthetic methods. Preparation of d-amino acids by deracemization has been conceptually attractive owing to facile synthesis of racemic amino acids by Strecker synthesis. Here, we demonstrated biocatalytic deracemization of aliphatic amino acids into d-enantiomers by running cascade reactions; (1) stereoinversion of l-amino acid to a d-form by amino acid dehydrogenase and ω-transaminase and (2) regeneration of NAD+ by NADH oxidase. Under the cascade reaction conditions containing 100?mM isopropylamine and 1?mM NAD+, complete deracemization of 100?mM dl-alanine was achieved after 24?h with 95% reaction yield of d-alanine (> 99% eeD, 52% isolation yield). Graphical Abstract: [Figure not available: see fulltext.].
Structure-guided engineering of: Meso -diaminopimelate dehydrogenase for enantioselective reductive amination of sterically bulky 2-keto acids
Cheng, Xinkuan,Chen, Xi,Feng, Jinhui,Wu, Qiaqing,Zhu, Dunming
, p. 4994 - 5002 (2018/10/17)
meso-Diaminopimelate dehydrogenase (DAPDH) and mutant enzymes are an excellent choice of biocatalysts for the conversion of 2-keto acids to the corresponding d-amino acids. However, their application in the enantioselective reductive amination of bulky 2-keto acids, such as phenylglyoxylic acid, 2-oxo-4-phenylbutyric acid, and indole-3-pyruvic acid, is still challenging. In this study, the structure-guided site-saturation mutagenesis of a Symbiobacterium thermophilum DAPDH (StDAPDH) gave rise to a double-site mutant W121L/H227I, which showed dramatically improved enzyme activities towards various 2-keto acids including these sterically bulky substrates. Several d-amino acids were prepared in optically pure form. The molecular docking of substrates into the active sites of wild-type and mutant W121L/H227I enzymes revealed that the substrate binding cavity of the mutant enzyme was reshaped to accommodate these bulky substrates, thus leading to higher enzyme activity. These results lay a foundation for further shaping the substrate binding pocket and manipulating the interactions between the substrate and binding sites to access highly active d-amino acid dehydrogenases for the preparation of synthetically challenging d-amino acids.
