327-57-1Relevant articles and documents
Enantioselective biocatalytic formal α-amination of hexanoic acid to l-norleucine
Dennig, Alexander,Gandomkar, Somayyeh,Cigan, Emmanuel,Reiter, Tamara C.,Haas, Thomas,Hall, Mélanie,Faber, Kurt
, p. 8030 - 8033 (2018)
A three-step one-pot biocatalytic cascade was designed for the enantioselective formal α-amination of hexanoic acid to l-norleucine. Regioselective hydroxylation by P450CLA peroxygenase to 2-hydroxyhexanoic acid was followed by oxidation to the ketoacid by two stereocomplementary dehydrogenases. Combination with final stereoselective reductive amination by amino acid dehydrogenase furnished l-norleucine in >97% ee.
Structural determinants for the non-canonical substrate specificity of the ω-transaminase from paracoccus denitrificans
Park, Eul-Soo,Park, Sae-Rom,Han, Sang-Woo,Dong, Joo-Young,Shin, Jong-Shik
, p. 212 - 220 (2014)
Substrate binding pockets of ω-transaminase (ω-TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω-TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω-TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω-TA whose S pocket shows a non-canonical steric constraint and readily accommodates up to an n-butyl substituent. The relaxed substrate specificity of the (S)-selective ω-TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as lnorvaline and l-norleucine. Molecular modeling using the recently released X-ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small-pocketforming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel-like structure allowing accommodation of linear alkyl groups of carboxylatebearing substrates. To verify the active site model, we introduced site-directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n-hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω-TA, which is expected to benefit rational redesign of substrate specificity of ω-TA.
2(S)-Aminohex-5-ynoic acid, an antimetabolite from Cortinarius claricolor var. Tenuipes
Aoyagi, Yasuo,Sugahara, Tatsuyuki
, p. 1835 - 1836 (1985)
Screening for antimetabolites in edible mushrooms showed that the hot water extract of fruiting bodies of Cortinarius claricolor var. tenuipes strongly inhibited the growth of Bacillus subtilis B-50 in a chemically defined minimal medium. 2(S)-Aminohex-5-ynoic acid was isolated as an active compound.
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.
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.
Preparative Asymmetric Synthesis of Canonical and Non-canonical a-amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
, p. 1348 - 1358 (2019/10/28)
Chemical and biocatalytic synthesis of non-canonical a-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-a-amino acids. In module 1, selective a-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable a-hydroxy acids (a-HAs; >90% a-selective) is shown on preparative scale (up to 2.3 gL1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of a-HAs into l-a-AAs (20 to 99%). Enantiopure l-a-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 gL1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs.
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.).
Preparative Asymmetric Synthesis of Canonical and Non-canonical α-amino Acids Through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
supporting information, (2019/02/09)
Chemical and biocatalytic synthesis of non-canonical α-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-α-amino acids. In module 1, selective α-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable α-hydroxy acids (α-HAs; >90% α-selective) is shown on preparative scale (up to 2.3 g L?1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of α-HAs into l-α-AAs (20 to 99%). Enantiopure l-α-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 g L?1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs. (Figure presented.).
Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system
Park, Eul-Soo,Shin, Jong-Shik
, p. 9 - 14 (2015/08/18)
Abstract Enantiopure aliphatic amino acids, including l-3-hydroxyadamantylglycine (l-Hag), l-tert-leucine (l-Tle) and l-norvaline, are essential chiral building blocks for a number of pharmaceutical drugs. Here, we developed cascade enzyme reactions in an extractive biphasic system using a branched-chain amino acid transaminase (BCTA) and an (S)-selective ω-transaminase (ω-TA) for asymmetric synthesis of the aliphatic amino acids from achiral α-keto acid precursors. The extractive cascade reactions enabled equilibrium shift of the BCTA reaction by recycling an amino acid cosubstrate as well as acceleration of the ω-TA reaction by removing an inhibitory ketone product from an aqueous phase. Starting with 20 mM α-keto acid, 4 mM rac-homoalanine and 50 mM rac-α-methylbenzylamine (rac-α-MBA), the biphasic cascade reactions afforded synthesis of four unnatural amino acids (i.e., l-Tle, l-Hag, l-norvaline and l-norleucine) and two natural amino acids (i.e., l-valine and l-Leucine) with >92% conversion yield and >99.9% ee. To demonstrate the industrial feasibility of the extractive cascade reaction, preparative-scale synthesis of l-Hag was performed in a reaction mixture consisting of 300 mL hexane and 50 mL aqueous solution (50 mM phosphate buffer, pH 7.0) charged with 50 mM keto acid substrate, 5 mM l-homoalanine, 120 mM rac-α-MBA, 2 U/mL BCTA and 16 U/mL ω-TA. Conversion yield of l-Hag reached 92% with >99.9% ee at 70 h. Product isolation led to 0.32 g white solid of l-Hag (62 % isolation yield).
Deracemization of amino acids by coupling transaminases of opposite stereoselectivity
Park, Eul-Soo,Shin, Jong-Shik
, p. 3505 - 3509 (2015/02/19)
Biocatalytic deracemization of amino acids without relying on oxidase-based deamination of an unwanted enantiomer was demonstrated by coupling a-and w-transaminases displaying opposite stereoselectivity. This strategy employs isopropylamine and a keto acid as cosubstrates and is free of generation of hydrogen peroxide which is troublesome in the conventional oxidase-based methods.
