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YggS is a pyridoxal 5'-phosphate (PLP)-binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP-dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP-dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5'-phosphate and α-keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

β-branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion and contemporary methods to achieve this often relies on the use of rare transition metal catalysis with designer ligand. Here, we report a biocatalytic transamination method to prepare a broad range of aromatic β-branched α-amino acids that proceeds with high diastereo- and enantioselectivity. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction is applied to the synthesis of several sp(3)-rich cyclic fragments and in the first total synthesis of jomthonic acid A.

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine., The use of continuous flow reactors enabled previously inaccessible enzyme combinations in multi‐enzyme cascade reactions. This included the combination of oxidases with aminating biocatalysts (transaminase/reductive aminase) and was extended to the continuous in situ biocatalytic generation of amino donors for the reductive aminase.

Metabolomics is one of the newest areas in biochemistry dedicated to investigating small biomolecules in biofluids, tissues, and cells. Cutting edge instruments used in metabolomics studies make it possible to identify thousands of biomolecules and determine their interactions with biological networks. This tremendous area has increased the significance of accurate chemical nomenclature of compounds. Therefore, the classification of the organic molecules has become one of the most important issues in the field. Biogenic amines are nitrogenous compounds of low molecular weight formed by the decarboxylation of amino acids or by the amination and the transamination of aldehydes and ketones during normal metabolic processes. This letter covers the topic of nomenclature with respect to the current usage of biogenic amines in scientific literature. We use metabolomics as an example of field reporting data on trace levels of molecules that may be miscategorized in primary literature. We suggest that the incorrect classification of molecules will influence science education adversely because resources used for teaching are drawn from primary literature references that may contain errors.

BACKGROUNDS The structural and interfacial properties of soybean protein isolate (SPI) after glycosylation by the transglutaminase method were studied. It is hoped that preliminary explorations will find a new food ingredient and broader application of SPI in the food industry. RESULTS The contents of free amino proves that transglutaminase can insert glucosamine into SPI through its transamination, and realize the enzymatic glycosylated SPI. The results of structure properties showed that a decrease in the content of the α-helical structure indicates that the rigid structure of the protein is opened and the flexibility is increased. The blue shift of the maximum fluorescence intensity of soy protein isolate-glucosamine with transglutaminase (SPI-G) indicates the formation of a new substance; scanning electron microscopy shows that the SPI-G powder can be seen at a magnification of 2000×, and the protein structure becomes soft. The results of interfacial properties found that enzymatic protein glycosylation exposes the internal hydrophobic groups of SPI, resulting in increased surface hydrophobicity, increased emulsification and emulsification stability, and reduced surface tension. CONCLUSION It shows that SPI-G effectively improves the interfacial properties of SPI, providing a theoretical basis for the application of enzymatic glycosylation of SPI in the food industry. © 2021 Society of Chemical Industry.

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α-carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.

We report a straightforward chemical strategy to tackle current challenges of irreversible deformation in low T-g vitrimers at operating temperature. In particular, vinylogous urethane (VU) vitrimers were prepared where reactive free amines, necessary for material flow, were temporarily shielded inside the network backbone, by adding a small amount of dibasic ester to the curing mixture. The amines could be released as reactive chain ends from the resulting dicarboxamide bonds via thermally reversible cyclisation to an imide moiety. Indeed, (re)generation of the required nucleophilic amines as network defects ensured reprocessing and rapid material flow at higher temperature, where exchange dynamics are (re)activated. As a result, VU vitrimers were obtained with limited creep at service temperature, yet with good reprocessability at elevated temperatures. Thus, by exerting strong control on the molecular level over the availability of exchangeable functional groups, a remarkable improvement of VU properties was obtained.

BACKGROUND: Amine transaminases have been extensively used for synthesizing various pharmaceutically relevant compounds, mainly in aqueous media. However, their applications are often limited by poor substrate solubility, low productivity and difficult product separation. This paper reports the use of Jeffamine® ED‐600, a novel polyether amine donor, for the transaminase‐catalyzed synthesis of 4‐phenyl‐2‐butylamine in non‐aqueous media. RESULTS: Enzymatic transamination was performed in the presence of a non‐polar organic solvent (n‐heptane), in which the selected amine donor is not soluble, thus a two‐liquid‐phase system was achieved. Coupling the reaction system with membrane‐assisted extraction resulted in simultaneous recovery of product, without any consistent contamination of the unreacted substrates. Moreover, a product yield of 60% was reached, compared with 15% without product extraction. The reaction was also successfully conducted without addition of any organic solvent, thus providing the first example of a solvent‐free transamination system. In the presence of only enzyme and substrates, up to 6‐fold higher product concentrations were achieved compared with the reaction performed in organic solvent. CONCLUSION: The use of the Jeffamine® ED‐600 in non‐aqueous media resulted beneficial for 4‐phenyl‐2‐butylamine synthesis. Enzymatic transamination in organic solvent with membrane‐assisted product extraction enabled shifting of the equilibrium and selective product extraction. Solvent‐free transamination minimized the required volume of the reactor and minimized the environmental impact. Extension to other substrate/enzyme solvent‐free systems could open new possibilities and perspectives in transaminase‐catalyzed chiral amine synthesis. © 2019 Society of Chemical Industry

Ni(II)-complexes, derived from glycine Schiff bases with chiral tridentate ligands, have been used as powerful tools for the synthesis of structurally diverse tailor-made amino acids. In this manuscript, asymmetric alkylation reaction between chiral nucleophilic glycine derived Ni-complex and 3-(chloromethyl)-1H-pyrrolo[2,3-b]pyridine has been developed under convenient conditions, which affords the corresponding alkylated Ni-complex in 74 % yield and excellent diastereoselectivity (only one isomer). This reaction features convenient conditions and completely controlled diastereoselectivity, which provides a highly valuable approach for asymmetric synthesis of 7-aza-tryptophan.

Pyridoxal 5'-phosphate (PLP)-dependent β-transaminases (βTAs) reversibly catalyze transamination reactions by recognizing amino groups linked to the β-carbon atoms of their substrates. Although several βTA structures have been determined as holo forms containing PLP, little is known about the effect of PLP on the conversion of the apo structure to the holo structure. We determined the crystal structure of the apo form of a βTA from Mesorhizobium sp. strain LUK at 2.2 Å resolution to elucidate how PLP affects the βTA structure. The structure revealed three major disordered regions near the active site. Structural comparison with the holo form also showed that the disordered regions in the apo form are ordered and partially adopt secondary structures in the holo form. These findings suggest that PLP incorporation into the active site contributes to the structural stability of the active site architecture, thereby forming the complete active site. Our results provide novel structural insights into the role of PLP in terms of active site formation.

R-ω-Transaminases (RTAs) catalyse the conversion of R-configured amines [e.g., (R)-1-phenylethylamine] into the corresponding ketones (e.g., acetophenone), by transferring an amino group from an amino donor [e.g., (R)-1-phenylethylamine] onto an amino acceptor (e.g., pyruvate), resulting in a co-product (e.g., d-alanine). d-Alanine can be deaminated back to pyruvate by d-amino acid oxidase (DAAOs). Here, through in vivo subunit splicing, the N terminus of an RTA subunit (RTAS ) was specifically ligated to the C terminus of a DAAO subunit (DAAOS ) through native peptide bonds (RTA&DAAO). RTAS is in close proximity to DAAOS , at a molecular-scale distance. Thus the transfer of pyruvate and d-alanine between RTA and DAAO can be directional and efficient. Pyruvate→d-alanine→pyruvate cycles are efficiently formed, thus promoting the forward transamination reaction. In a different, in vitro noncovalent approach, based on coiled-coil association, the RTAS N terminus was specifically associated with the DAAOS C terminus (RTA#DAAO). In addition, the two mixed individual enzymes (RTA+DAAO) were also studied. RTA&DAAO has a shorter distance between the paired subunits (RTAS -DAAOS ) than RTA#DAAO, and the number of the paired subunits is higher than in the case of RTA#DAAO, whereas RTA+DAAO cannot form the paired subunits. RTA&DAAO exhibited a transamination catalysis efficiency higher than that of RTA#DAAO and much higher than that of RTA+DAAO.

An unprecedented, efficient I2-mediated approach for the synthesis of 6-phenylpyrido[2',1':2,3]imidazo[4,5-c]quinolone heterocycle skeletons has been achieved from benzylamines and 2-(imidazo[1,2-a]pyridin-2-yl)aniline. This protocol proceeds via a sequential oxidation/transamination/cyclization/aromatisationin one pot resulting in the formation of new N-C and C-C bonds under metal-free conditions. A variety of 2 benzylamines including pyridin-2-ylmethanamine were successfully utilized as an imine precursors and employed in irreversible transamination with 2-(imidazo[1,2-a]pyridin-2-yl)anilines to generate the corresponding imines, which subsequently cyclized to corresponding 6-phenylpyrido[2',1':2,3]imidazo[4,5-c]quinolone under the optimised conditions.

International audience; We described an efficient in situ generation of hydroxypyruvate from d‐serine catalyzed by a d‐amino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l‐serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbon−carbon bond. The enzymatic cascade reaction was performed in one‐pot in the presence of d‐serine and appropriate aldehydes for the synthesis of valuable (3S)‐hydroxyketones, which were obtained with high enantio‐ and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d‐serine and aldehydes.

As a complementary approach to current asymmetric syntheses, a direct transformation of styrenes into the corresponding 1-phenylethylamines was developed. The one-pot process represents a formal hydroamination of a non-activated alkene with ammonia and is based on the combination of a Pd/Cu-catalyzed Wacker-oxidation with an enzymatic transamination. A key feature of this process is the compartmentation of the catalytic systems, which turned out to be essential due to deactivation of the transaminase by the Cu-component. This one-pot synthesis can be applied for a broad substrate range leading to the chiral amines with high conversions and excellent enantioselectivities of >99% ee.

The transamination of 6-chloro-2-(3-chlorophenyl)-1H-benz[de]isoquinoline-1,3-(2H)-dione with primary alkylamines under pseudo-first-order conditions is shown to be unimolecular in both reagents as well as unimolecular in methanol (in methanol solution). The reaction is subject to both general acid and general base catalysis. A termolecular, solvated transition state model and a putative model for the early steps of laser tissue welding with aminonaphthalimide dyes are proposed.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-1 h-1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc.

Key points Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney. Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation. Abstract The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl-l-histidine) in muscle. However, only a small fraction (3–6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake.

Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product has led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidation of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications in order to accommodate aldehyde-containing substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chemically or enzymatically to introduce aldehyde functional groups. Finally, the total chemical synthesis of proteins complements these biological and chemoenzymatic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivatives to tune the activity, pH preference, stability and solubility of the catalyst. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

Just like Nature: A recently developed enantioselective organocatalytic biomimetic transamination provides an elegant approach towards chiral amines. In the presence of an asymmetric phase-transfer catalyst, the intermediate anionic species undergoes an asymmetric C-C bond-forming reaction in a powerful and broadly applicable asymmetric umpolung strategy.

The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM = 54.1 µM-1 Min-1 (KM = 32 ± 3 µM; kcat = 1,730 ± 39 Min-1 ), almost the same as the wild-type enzyme for NADH (kcat /KM = 59.9 µM-1 Min-1 ; KM = 14 ± 2 µM; kcat = 838 ± 21 Min-1 ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM = 3 µM-1 Min-1 ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP+ and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP+ and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used to treat life-threatening bacterial infections. The gentamicin C complex consists of gentamicin C1, gentamicin C1a, and epimers gentamicin C2 and gentamicin C2a. At present there is a generally accepted pathway of gentamicin biosynthesis, except for detailed understanding of the epimerization process involving gentamicins C2 and C2a. Here we have investigated the biosynthesis of these epimers. JI-20B-an intermediate in the gentamicin biosynthetic pathway-and its epimer JI-20Ba were generated by in-frame deletion within genP, which encodes a phosphotransferase that catalyzes the first step of 3',4'-bisdehydroxylation in gentamicin biosynthesis. GenB1 and GenB2 are aminotransferases with different substrate specificities and enantioselectivities. JI-20Ba, containing a 6'S chiral amine, a precursor of gentamicin C2a, was synthesized from G418 by GenQ/GenB1 through sequential oxidation/transamination at C-6'. GenQ/GenB2 catalyzed the synthesis of JI-20B, containing a 6'R chiral amine, a precursor of gentamicin C2, from G418. GenB2 catalyzed the epimerization of JI-20Ba/JI-20B and of gentamicins C2a/C2.

The reaction of 4-aminocoumarin (2) with benzaldehyde gave the bisenamino derivatives 4. Thus, transamination of 2 with o-phenylenediamine furnished enamino skeleton 3, which can be obtained from 1. Reaction of 1 with 5-aminoisoxazole afforded 5. While reaction of 2 with benzalaniline, isatinanil and phenyl isothio cyanate afforded the corresponding 7, 8 and 9, respectively. Heterocyclic annulations of 2-phenylthiazolidine-4-one 10 2-phenylthiazolidin-4-one 10 and 2-phenyl-thiazinan-4-one 11 systems were achieved via reaction of 2 with benzaldehyde and rather either 2-mercaptoacetic acid or 3-mercaptopropanoic acid respectively. The behavior of 2 towards monochloro acetic acid and 4-oxo-4-phenylbuatonic 4-Oxo-4-phenylbutanoic acid was investigated. All of the newly synthesized compounds were evaluated as antitumor (cytotoxic) agents. Most of these compounds have shown significant antitumor activities.

Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of a-hydroxy ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30-fold increased activity in the conversion of (S)-phenylbutylamine and (R)-phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98% ee. QC 20150408

International audience; The direct oxidative cross-coupling of primary amines is a challenging transformation as homocoupling is usually preferred. We report herein the chemoselective preparation of cross-coupled imines through the synergistic combination of low loadings of Cu II metal-catalyst and o-iminoquinone organocatalyst under ambient conditions. This homogeneous cooperative catalytic system has been inspired by the reaction of copper amine oxidases, a family of metalloenzymes with quinone organic cofactors that mediate the selective oxidation of primary amines to aldehydes. After optimization, the desired cross-coupled imines are obtained in high yields with broad substrate scope through a transamination process, that leads to the homocoupled imine intermediate, followed by dynamic transimination. The ability to carry out the reactions, at room temperature, with ambient air rather than molecular oxygen as the oxidant and equimolar amounts of each coupling partner, is particularly attractive from an environmentally viewpoint.

Metabolic engineering of microbial strains to produce aromatic compounds deriving from the shikimate pathway is of great interest to the chemical industry as a more sustainable alternative for feedstock production. Chorismate is a significant intermediate in the shikimate pathway. In this study, the formation of phenylalanine and phenylpyruvate as by-products in strains engineered downstream of the chorismate node for increased aromatic production was explored in yeast fermentations. Tracer experiments showed that these compounds are synthesized de novo during fermentation, under conditions in which their synthesis was genetically blocked. Chorismate stability evaluation, as well as deletion mutation analysis throughout the phenylalanine biosynthesis pathway, suggested that this synthesis was a result of intracellular, non-enzymatic rearrangement of chorismate to phenylpyruvate via prephenate, which was followed by enzymatic transamination of phenylpyruvate to form phenylalanine. These results not only aid in the development of strain-engineering strategies to avoid the accumulation of by-products during fermentations aimed at increased aromatics production, but also deepen our understanding of yeast metabolism.

The importance and structural diversity of chiral amines is well-demonstrated by the myriad nonenzymatic methods for their chemical production. In nature, the production of amines is performed by transamination rather than by reduction of an imine precursor derived from the corresponding ketone. Imine reductases, however, show great potential in the reduction of cyclic imines that are stable towards hydrolysis in aqueous reaction media. Here, we report the catalytic activity of two S-selective imine reductases towards 3,4-dihydroisoquinolines and 3,4-dihydro-β-carbolines and their activity in the direct reductive amination of ketone substrates. The crystal structures of the enzyme from Streptomyces sp. GF3546 in complex with the cofactor NADPH and from Streptomyces aurantiacus in native form have been solved and refined to a resolution of 1.9 A.

A new series of tricyclic pyrimidoquinoxaline derivatives were synthesized and evaluated as antitumor assays and compared with standard drug 5-fluorouracil. These new pyrimidoquinoxaline derivatives were synthesized by the reaction with o-aminonitrilequinoxaline derivative 3 with various reagents. One from which, the condensation of o-aminonitrile with potassium cyanate in acetic acid was stated as a new procedure for building the pyrimidine ring incorporate to quinoxaline moiety. Further condensation of aminonitrile 3 with formamide or Vilsmeier reaction followed by transamination or carbon disulphide was applied as procedures for the pyrimidine ring syntheses. Compound 15 achieved significant in vitro antitumor activity, and compounds 9 and 14 have high activities.

Malassezia yeasts are responsible for the widely distributed skin disease Pityriasis versicolor (PV), which is characterized by a hyper- or hypopigmentation of affected skin areas. For Malassezia furfur, it has been shown that pigment production relies on tryptophan metabolism. A tryptophan aminotransferase was found to catalyse the initial catalytic step in pigment formation in the model organism Ustilago maydis. Here, we describe the sequence determination, recombinant production and biochemical characterization of tryptophan aminotransferase MfTam1 from M. furfur. The enzyme catalyses the transamination from l-tryptophan to keto acids such as α-ketoglutarate with Km values for both substrates in the low millimolar range. Furthermore, MfTam1 presents a temperature optimum at 40°C and a pH optimum at 8.0. MfTam1 activity is highly dependent on pyridoxal phosphate (PLP), whereas compounds interfering with PLP, such as cycloserine (CS) and aminooxyacetate, inhibit the MfTam1 reaction. CS is known to reverse hyperpigmentation in PV. Thus, the results of the present study give a deeper insight into the role of MfTam1 in PV pathogenesis and as potential target for the development of novel PV therapeutics.

2-Phenylethanol (2-PE), a fragrance compound with a rose-like odor, is widely used in perfumery and cosmetics. Here, we report the first metabolic engineering approach for 2-PE production in Saccharomyces cerevisiae. 2-PE can be produced from the catabolism of L-phenylalanine via Ehrlich pathway, consisting of transamination to phenylpyruvate by Aro9, decarboxylation to phenylacetaldehyde by Aro10, and reduction to 2-PE by alcohol dehydrogenases. We demonstrated that Ald3 is mainly responsible for phenylacetaldehyde oxidation, competing with 2-PE production. ALD3 deletion strain overexpressing ARO9 and ARO10 both by episomal overexpression and by induction of the endogenous genes through overexpression of Aro80 transcription factor, produced 4.8 g/L 2-PE in a medium containing 10 g/L L-phenylalanine as a sole nitrogen source. Considering the cytotoxicity of 2-PE, this production titer is almost the upper limit that can be reached in batch cultures, suggesting the great potential of this yeast strain for 2-PE production. 2-PE production was further increased by applying two-phase fermentation method with polypropylene glycol 1200 as an extractant, reaching 6.1 g/L 2-PE in organic phase with the molar yield of 82.5%, which is about ninefold increase compared with wild type.

Syntheses and structures of five imido-bridged dinuclear titanium complexes and two (bis)ligand-coordinated mononuclear titanium complexes are reported. Addition of 1 or 2 equiv. of Schiff base ligand (((1H-pyrrol-2-yl)methylene)amino)-2,3-dihydro-1H-inden-2-ol (H2L) to Ti(NMe2)4 resulted in transamination with 4 equiv. of dimethylamides generating a (bis)ligand-coordinated complex Ti(L)2 (1). Treatment of Ti(NMe2)4 with 1 equiv. of tBuNH2 followed by addition of 1 equiv. of H2L afforded an imido-bridged complex [Ti(L)(NtBu)]2 (2). 1:1:1:1 reaction of Ti(NMe2)4/RNH2/H2L/py(or phen) produced imido-bridgedcomplexes [Ti(L)(NPh)(py)]2 (3), [Ti(L)(4-F-PhN)(py)]2·Tol (4·Tol), [Ti(L)(4-Cl-PhN)(py)]2·Tol·THF (5·Tol·THF), [Ti(L)(4-Br-PhN)(py)]2·Tol (6·Tol) and a (bis)ligand-coordinated complex Ti(L)2·phen (7) (py = pyridine, phen = 1,10-phenanthroline). Attempts to prepare the monomeric titianium imido complexes were unsuccessful. DFT studies show that the assumed compound which contains Ti = N species is less stable than imido-bridged Ti-N(R)-Ti complexes, providing the better understanding of the experimental results.

Apart from their crucial role in metabolism, pyridoxal 5'-phosphate (PLP)-dependent aminotransferases (ATs) constitute a class of enzymes with increasing application in industrial biotechnology. To provide better insight into the structure-function relationships of ATs with biotechnological potential we performed a fundamental bioinformatics analysis of 330 representative sequences of pro- and eukaryotic Class III ATs using a structure-guided approach. The calculated phylogenetic maximum likelihood tree revealed six distinct clades of which the first segregates with a very high bootstrap value of 92%. Most enzymes in this first clade have been functionally well characterized, whereas knowledge about the natural functions and substrates of enzymes in the other branches is sparse. Notably, in those clades 2-6 members of the peculiar class of ω-ATs prevail, many of which have proven useful for the preparation of chiral amines or artificial amino acids. One representative is the ω-AT from Paracoccus denitrificans (PD ω-AT) which catalyzes, for example, the transamination in a novel biocatalytic process for the production of L-homoalanine from L-threonine. To gain structural insight into this important enzyme, its X-ray analysis was carried out at a resolution of 2.6 A, including the covalently bound PLP as well as 5-aminopentanoate as a putative amino donor substrate. On the basis of this crystal structure in conjunction with our phylogenetic analysis, we have identified a generic set of active site residues of ω-ATs that are associated with a strong preference for aromatic substrates, thus guiding the discovery of novel promising enzymes for the biotechnological production of corresponding chiral amines.

A new type of novel chiral pyridoxamines 3a–g containing a side chain has been developed. The pyridoxamines displayed catalytic activity and promising enantioselectivity in biomimetic asymmetric transamination of α-keto acids, to give various α-amino acids in 47–90% yields with up to 87% ee’s under very mild conditions. An interesting effect of the side chain on enantioselectivity was observed in the reaction.

The reaction of 4-aminocoumarin (2) with benzaldehyde gave the bisenamino derivatives 4. Thus, transamination of 2 with o-phenylenediamine furnished enamino skeleton 3, which can be obtained from 1. Reaction of 1 with 5-aminoisoxazole afforded 5. While reaction of 2 with benzalaniline, isatinanil and phenyl isothio cyanate afforded the corresponding 7, 8 and 9, respectively. Heterocyclic annulations of 2-phenylthiazolidine-4-one 10 2-phenylthiazolidin-4-one 10 and 2-phenyl-thiazinan-4-one 11 systems were achieved via reaction of 2 with benzaldehyde and rather either 2-mercaptoacetic acid or 3-mercaptopropanoic acid respectively. The behavior of 2 towards monochloro acetic acid and 4-oxo-4-phenylbuatonic 4-Oxo-4-phenylbutanoic acid was investigated. All of the newly synthesized compounds were evaluated as antitumor (cytotoxic) agents. Most of these compounds have shown significant antitumor activities.

2,2-Dinitroethene-1,1-diamine (FOX-7) is an insensitive, energetic material with a simple structure but very unusual properties. With interest among chemists in expanding the chemistry of FOX-7, continuous efforts are underway to improve the performance of the compound by modifying and optimizing its structure and creating various derivatives. In this Review, the chemical reactivity of 1,1-diamino-2,2-dinitroethene (FOX-7) towards nucleophilic (transamination), and electrophilic substitution, and its acid–base properties are explored as well as an account of the molecular structure, physical and detonation properties of derivatives, salts and metal complexes of FOX-7.

The title sequence starts from β,γ-unsaturated α-ketoesters and 1,3-cyclohexanediones in the presence of the optically active ligand DASQ.

Cystathionine γ-synthase (CGS) catalyzes the condensation of O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce L-cystathionine (L-Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the futile α,γ-elimination of L-OSHS, yielding succinate, α-ketobutyrate, and ammonia. A series of 16 site-directed variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the α,γ-elimination reaction range from a reduction of only ∼2-fold for R49K and the E325A,Q variants to 310- and 760-fold for R361K and R48K, respectively. A similar trend is observed for the k(cat) /K(m)(l-OSHS) of the physiological, α,γ-replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and α-carboxylate moieties, respectively, of the L-OSHS substrate. In contrast, with the exception of the 13-fold increase observed for R106A, the K(m)(l-Cys) is not markedly affected by the site-directed replacement of the residues investigated. The decrease in k(cat) observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active-site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L-Ala.

The equilibrium constant is a critical parameter for making rational design choices in biocatalytic transamination for the synthesis of chiral amines. However, very few reports are available in the scientific literature determining the equilibrium constant (K) for the transamination of ketones. Various methods for determining (or estimating) equilibrium have previously been suggested, both experimental as well as computational (based on group contribution methods). However, none of these were found suitable for determining the equilibrium constant for the transamination of ketones. Therefore, in this communication we suggest a simple experimental methodology which we hope will stimulate more accurate determination of thermodynamic equilibria when reporting the results of transaminase-catalyzed reactions in order to increase understanding of the relationship between substrate and product molecular structure on reaction thermodynamics. Biotechnol. Bioeng. 2012; 109:2159–2162. © 2012 Wiley Periodicals, Inc.

The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, ...

Summary In the present study, two freshwater fishes Channa punctatus and Clarias batrachus were exposed to sub-acute concentrations of a commercial-grade k-cyhalothrin, REEVA-5, for 96 h to observe the changes in amino acid catabolism under pyrethroidinduced stress and to investigate the comparative mechanisms of ammonia detoxification in both fishes. The experiments included the estimation of levels of free amino acid, urea, ammonia and the specific activities of aspartate aminotransferase (AAT), alanine aminotransferase (AlAT), glutamate dehydrogenase (GDH), glutamine synthetase (GS) and arginase in different vital organs of fishes. k-cyhalothrin caused significant decline in the levels of amino acids along with simultaneous significant increase in the activity of AAT, AlAT and GDH, which indicated amino acid catabolism as one of the important mechanisms to meet out immediate energy demand of fishes. The level of ammonia was observed to be enhanced considerably at lower concentrations of k-cyhalothrin while higher concentrations caused remarkable decline. The k-cyhalothrin treatment resulted in significant increase in the activities of GDH and GS with concomitant increase in the activity of arginase and level of urea, indicating activation of two different mechanisms of ammonia detoxification. The mechanism of ammonia detoxification through its conversion into glutamate and glutamine was more prominent in C. punctatus, while C. batrachus demonstrated ureogenesis as the major route. In fishwise comparison, C. batrachus was observed to be more sensitive with respect to the above-mentioned parameters. Another important finding was that unlike the liver, the kidney appeared as one of the primary sites of ureogenesis in fishes.

Biomimetic studies of pyridoxal and pyridoxamine models are of both fundamental and practical interest. This review examines (i) deracemization of α-amino acids with a chiral pyridoxal model, (ii) sensing of chirality of small molecules including α-, β-, and γ-amino acids, peptides, amino alcohols and diamines with an achiral pyridoxal model and (iii) stereospecific synthesis of chiral diamines with a chiral pyridoxamine model. A binol-based aldehyde is useful as a chiral pyridoxal model to deracemize a variety of α-amino acids to make D-amino acids. 2,2′-Dihydroxybenzophenone is useful as an achiral pyridoxal model for sensing the chirality of the above-mentioned small molecules in a unified way. Bis(2-hydroxyphenyl)-ethylenediamine (hpen) is useful as a chiral pyridoxamine model for making chiral diamines. Weak forces (H-bonding, steric and electronic effects) involved in transamination reactions with pyridoxamine for the synthesis of amino acids are compared with those for the diaza-Cope rearrangement reaction with hpen for the synthesis of chiral diamines. Both experimental and computational methods are used to analyze the biomimetic systems.

Cyanoacylation of 2-amino-tetrahydrobenzothiophene-3-carboxylate ethyl ester with 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile afforded cyanoacetamide 2. The later was utilized as key intermediate for the synthesis of 3-substituted 2-iminocoumarins 3, 4, 5, 6 and acrylamides 7a, b via Knoevenagel condensation with 2-hydroxy-1-naphthaldehyde; 2-hydroxybenzaldehyde; 1-nitrosonaphthalen-2-ol; 7-hydroxy-5-methoxy-2-methyl-4-oxo-4H-chromene-6-carbaldehyde; 4-dimethylamino-benzaldehyde; and 4-piperidin-1-yl-benzaldehyde in EtOH/piperidine. The derivatives 7a, b did not afford the pyrazoles 8a, b upon treating with phenyl hydrazine. Furthermore, coupling of 2 with 4-amino-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one and 4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-amine afforded the hydrazone derivatives 9 and 10, respectively. The later derivative 10 was cyclized in acetic acid to afford the pyridopyrazolotriazine 11. Finally, 2 was treated with dimethylformamide-dimethylacetal (DMF-DMA) to afford the dimethylaminoacrylamide 12 which underwent transamination with 4,6-dimethyl-1H-pyrrolo[2,3-b]pyridin-3-amine to afford the pyrazole 13. Cyclization of compound 13 in acetic acid or pyridine was unsuccessful. The antitumor and antioxidant activities of the synthesized products were evaluated; several were found to exhibit promising antioxidant activities. J. Heterocyclic Chem., (2011).