Exploring the Active Site of Amine:Pyruvate Aminotransferase on the Basis of the Substrate Structure−Reactivity Relationship:  How the Enzyme Controls Substrate Specificity and Stereoselectivity

Shin, Jaeyoung; Kim, Byung Gee

doi:10.1021/jo016115i

Cited by 152 publications

(137 citation statements)

References 12 publications

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“…2). The relative apparent activities of the crude cell extract were always in good agreement with previous results obtained for the purified enzyme (14). Compared with wild-type -TA, the -TAmla crude extract displayed twofold-higher activity for long-chain aliphatic amines, such as 2-aminoheptane (A4), 1,5-dimetylhexylamine (A5), and 1-methylheptylamine (A6) in the reaction conditions used (Fig.…”

Section: Vol 71 2005 Directed Evolution Of V Fluvialis Js17 -Ta 4221supporting

confidence: 89%

“…In E. coli BL21 cells harboring pET24ma with and without the -TA gene, only the cells with the insert grew. Compared to the activity with (S)-␣-MBA, the apparent activities of -TA with 2-aminoheptane and 2-aminobutane are 43 and 7%, respectively (14), indicating that 2-aminoheptane is a better amino group donor than 2-aminobutane. This was reflected in the growth pattern of the recombinant E. coli possessing -TA activity, which grew three times faster in the minimal medium containing 2 mM 2-aminoheptane than in the minimal medium containing 2 mM 2-aminobutane.…”

Section: Resultsmentioning

confidence: 87%

“…The -TA from Vibrio fluvialis JS17 screened from soil microorganism shows high enantioselectivity (E) for the (S) enantiomers of various chiral amines, such as ␣-MBA and secbutylamine, with remarkable stability and a high reaction rate (11)(12)(13)(14)(15)(16). However, severe inhibition by the ketone product prevented the enzyme from maintaining the maximum activity.…”

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confidence: 99%

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Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Yun

Hwang

Lee

et al. 2005

Appl Environ Microbiol

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A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant -transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, -TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6-and 4.5-fold higher than those of the wild type, respectively. Using -TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

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Section: Vol 71 2005 Directed Evolution Of V Fluvialis Js17 -Ta 4221supporting

confidence: 89%

Section: Resultsmentioning

confidence: 87%

mentioning

confidence: 99%

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Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Yun

Hwang

Lee

et al. 2005

Appl Environ Microbiol

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“…It is generally accepted that both (R)-and (S)-selective -TAs possess two substrate-binding pockets consisting of a large pocket that is capable of dual recognition of hydrophobic and carboxyl groups and a small pocket that can accept substituents no larger than an ethyl group (27,28). The canonical structural features of the active site of -TAs render a carboxyl group and a side chain of keto acid substrates bound to the large and small pockets, respectively.…”

mentioning

confidence: 99%

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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-Transaminase (-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most -TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective -TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for ␣-keto acids and ␣-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48-and 56-fold increases in activity for 2-oxopentanoic acid and L-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an ␣-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure L-and D-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. U nnatural amino acids are widely used as essential chiral building blocks for diverse pharmaceutical drugs, agrochemicals, and chiral ligands (1-3). In contrast to natural amino acids, a fermentative method for the production of unnatural amino acids is not yet commercially available (4,5). This has driven the development of various biocatalytic approaches to afford scalable processes for preparing enantiopure unnatural amino acids. These processes include kinetic resolution of racemic amino acids using acylase (6, 7), amidase (8, 9), hydantoinase (10, 11), and amino acid oxidase (12, 13) and asymmetric reductive amination of keto acids using dehydrogenase (14, 15) and transaminase (16,17). Asymmetric amination is usually favored over kinetic resolution because the former affords a 100% yield without racemization of an unwanted enantiomer.In contrast to a mandatory requirement for the supply and regeneration of an expensive external cofactor for the dehydrogenase reactions, transaminase catalyzes the transfer of an amino group from an amino donor to an acceptor using pyridoxal 5=-phosphate (PLP) as a prosthetic group (18). Nevertheless, industrial implementation of the transaminase-mediated synthesis of unnatural amino acids has lagged behind because of low equilibrium constants, usually close to unity, for the reactions between keto acids a...

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“…This is similar to !-ATVf, which shows relatively low activity towards aromatic primary amine compounds (19.7-23.4%) and for aliphatic amines (1.7-18.5%) as compared to the activity towards (S)--methylbenzylamine. 36) Although mlr6963 and mll3663 showed similar scores on profile analysis, mlr6963 showed broad substrate specificity for all the substrates examined, whereas mll3663 showed narrow specificity towards aromatic amines, such as A1 and A2, and low activities towards B4 and B6. Mll5987 showed higher activity for amines than for -amino acids.…”

Section: Experimental Investigation Of the Functions Of The Putative mentioning

confidence: 91%

Computational Selection, Identification and Structural Analysis of ω-Aminotransferases with Various Substrate Specificities from the Genome Sequence ofMesorhizobium lotiMAFF303099

Seo

Hwang

Seo

et al. 2012

Bioscience, Biotechnology, and Biochemistry

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Exploring the Active Site of Amine:Pyruvate Aminotransferase on the Basis of the Substrate Structure−Reactivity Relationship: How the Enzyme Controls Substrate Specificity and Stereoselectivity

Cited by 152 publications

References 12 publications

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Computational Selection, Identification and Structural Analysis of ω-Aminotransferases with Various Substrate Specificities from the Genome Sequence ofMesorhizobium lotiMAFF303099

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