Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

Patel, Ramesh N.

doi:10.3390/biom3040741

Cited by 116 publications

(70 citation statements)

References 129 publications

(163 reference statements)

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“…In addition to its activity with allylic aldehydes, the organism can reduce acetophenone and its derivatives to chiral aromatic alcohols, some of which are valuable intermediates for synthesis of biologically or pharmacologically active compounds (29). Bioreduction catalyzed by whole cells of Yokenella sp.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Ying

Wang

Xiong

et al. 2014

Appl Environ Microbiol

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A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the CAO bond of allylic aldehydes/ketones to the corresponding ␣,␤-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg ؊1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg ؊1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP ؉ , suggesting the nature of being an aldehyde reductase.

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Section: Discussionmentioning

confidence: 99%

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Ying

Wang

Xiong

et al. 2014

Appl Environ Microbiol

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“…Such noncanonical amino acids (NCAAs) have diverse applications in chemical biology (8), serve as intermediates in the synthesis of natural products (9,10), and are privileged scaffolds for the development of pharmaceuticals (11). Despite its natural ability to produce these desirable compounds, TrpS has enjoyed only limited application (7).…”

mentioning

confidence: 99%

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Buller

Brinkmann‐Chen

Romney

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

146

257

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Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.protein engineering | allostery | noncanonical amino acid | PLP

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“…nnatural amino acids are widely used as essential chiral building blocks for diverse pharmaceutical drugs, agrochemicals, and chiral ligands (1)(2)(3). In contrast to natural amino acids, a fermentative method for the production of unnatural amino acids is not yet commercially available (4,5).…”

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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Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

Cited by 116 publications

References 129 publications

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

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

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