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- Title
Active site model of ( R)-selective ω-transaminase and its application to the production of d-amino acids.
- Authors
Park, Eul-Soo; Dong, Joo-Young; Shin, Jong-Shik
- Abstract
ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of l-amino acids using ( S)-selective ω-TAs. However, despite the availability of ( R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of d-amino acids. Here, we investigated the substrate specificity of ( R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of d-amino acids from α-keto acids. Substrate specificity toward d-amino acids and α-keto acids revealed that the two ( R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of ( S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized ( S)-selective ω-TAs, the enzyme activity of the ( R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five d-amino acids ( d-alanine, d-homoalanine, d-fluoroalanine, d-serine, and d-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7 %).
- Subjects
AMINOTRANSFERASES; ENZYME specificity; AMINO acid analysis; ENZYME activation; ACETOPHENONE; CHEMICAL sample preparation
- Publication
Applied Microbiology & Biotechnology, 2014, Vol 98, Issue 2, p651
- ISSN
0175-7598
- Publication type
Academic Journal
- DOI
10.1007/s00253-013-4846-5