Enzymatic Synthesis of L-tert-Leucine with Branched Chain Aminotransferase

Seo, Young-Man; Yun, Hyungdon

doi:10.4014/jmb.1105.05049

Cited by 19 publications

(13 citation statements)

References 16 publications

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“…Such information would be of importance for evaluating process technology options [10]. Earlier studies [11][12][13][14][15] had alerted us to the potential of BCAT as a biocatalyst for production of high-value non-natural amino acids, such as L-tert-leucine and L-3-hydroxyadamantylglycine. Therefore, the overall substrate specificity and kinetic properties of E. coli BCAT have been revisited using the novel coupled assay, which delivers reliable initial-rate measurements.…”

Section: Introductionmentioning

confidence: 99%

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Wang

Engel

2013

The FEBS Journal

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Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/ acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as L-norleucine, L-norvaline and L-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steadystate kinetic study for several substrates yields results consistent with a bibi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation. DatabaseBranched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2

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Section: Introductionmentioning

confidence: 99%

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Wang

Engel

2013

The FEBS Journal

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“…Previous studies have reported L -tert-leucine synthesis using BCAT/AspAT/PDC, BCAT/ω-AT, and BCAT/GluDH/FDH coupling systems. In the BCAT/AspAT/PDC coupling system, the formation of L -glutamic acid from α-ketoglutaric acid was favoured by the spontaneous decarboxylation of oxaloacetate to form pyruvate (16). Pyruvate decarboxylase (PDC) further performs the non-oxidative thiamine diphosphate-mediated decarboxylation of pyruvate to shift the equilibrium of the reaction (16).…”

Section: Discussionmentioning

confidence: 99%

“…In the BCAT/AspAT/PDC coupling system, the formation of L -glutamic acid from α-ketoglutaric acid was favoured by the spontaneous decarboxylation of oxaloacetate to form pyruvate (16). Pyruvate decarboxylase (PDC) further performs the non-oxidative thiamine diphosphate-mediated decarboxylation of pyruvate to shift the equilibrium of the reaction (16). The BCAT/ω-AT coupling reaction was also developed to eliminate the product inhibition by simultaneously performing an energetically favourable ω-AT reaction using a two-liquid phase reaction system (17).…”

Section: Discussionmentioning

confidence: 99%

“…The biosynthesis of L -tert-leucine has been achieved by coupling BCAT with several enzymes, including ornithine-δ-aminotransferase (OAT) (14), AspAT/pyruvate decarboxylase (PDC) (15), ω-AT (16), and GluDH/FDH (17). However, the crucial setbacks in the scale-up of the most BCTA coupling reaction include low catalytic efficiency, cumbersome steps and high cost.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Zheng

Cui

et al. 2019

Preprint

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Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

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“…For the first time, the microbial synthesis of these products benefitted from a strong support of the interlinked carbon core metabolism. In contrast to the previously tested host E. coli (Hong et al, 2010;Hwang et al, 2011;Seo and Yun, 2011), C. glutamicum efficiently supplied glutamate as amino group donor for the biotransformation and, at the same time, removed the enzyme inhibitor a-ketoglutarate via its endogenous path-ways. Accordingly, the reaction could be realized as a one-pot process.…”

Section: Discussionmentioning

confidence: 99%

Production of non‐proteinogenic amino acids from α‐keto acid precursors with recombinant Corynebacterium glutamicum

Kim

Lee

Wittmann

et al. 2013

Biotech & Bioengineering

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In the present work, Corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. The novel bio-catalytic activity of C. glutamicum was obtained by heterologous expression of the branched chain aminotransferase IlvE from Escherichia coli. Upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (HOAE) into the corresponding amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid (HAAE). Similarly, also L-tert-leucine could be obtained from trimethyl pyruvate indicating a broader applicability of the novel strategy. In both cases, the amino group donor glutamate was supplied from the endogenous metabolism of the recombinant producer. Hereby, the uptake of the precursor and secretion of the product was supported by an enhanced cell permeability through treatment of ethambutol, which inhibits arabinosyl transferases involved in cell wall biosynthesis. The excretion of HAAE into the reaction medium was linked to the secretion of glutamate, indicating a similar mechanism for the export of both compounds. On the other hand, the efflux of L-tert-leucine appeared to be driven by active transport. Subsequent bioprocess engineering enabled HAAE and L-tert-leucine to be produced at a rate of 0.21 and 0.42 mmol (g dry cells)⁻¹ h⁻¹, respectively up to a final product titer of 40 mM. Beyond the given examples, integrated metabolic and cell envelop engineering might extend the production of a variety of other non-proteinogenic amino acids as well as chiral amines by C. glutamicum.

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Enzymatic Synthesis of L-tert-Leucine with Branched Chain Aminotransferase

Cited by 19 publications

References 16 publications

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Production of non‐proteinogenic amino acids from α‐keto acid precursors with recombinant Corynebacterium glutamicum

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