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Applied Microbiology and Biotechnology

, Volume 103, Issue 19, pp 8051–8062 | Cite as

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

  • Xinxin Zheng
  • Yinglu Cui
  • Tao Li
  • Ruifeng Li
  • Lu Guo
  • Defeng Li
  • Bian WuEmail author
Biotechnologically relevant enzymes and proteins
  • 148 Downloads

Abstract

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. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can use branched-chain amino acids (BCAAs) as substrate, including L-valine, L-leucine, and L-isoleucine, with α-ketoglutarate to form the corresponding α-keto acids and L-glutamate. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine and L-norvaline. 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 bulked aliphatic L-amino acids (kcat up to 220 s−1). Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. To analyze the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. 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. Finally, conjugated with the ornithine aminotransferase (OrnAT) to shift the reaction equilibrium towards the product formation, the coupled system was applied to the asymmetric synthesis of L-tert-leucine and L-norvaline. In summary, the structural and functional characteristics of PsBCAT were analyzed in detail, and this information will be conducive to industrial production of enantiopure chiral amino acids by aminotransferase.

Keywords

Branched-chain amino acid aminotransferase Pseudomonas sp. crystal structure Chiral amino acids 

Notes

Acknowledgments

We would like to thank Juncai Ma and Linhuan Wu from Institute of Microbiology, Chinese Academy of Sciences, for submitting our sequence to the GCM Type strain genome database.

Funding information

This work is supported by the National Key R&D Program of China (Grant No. 2018YFA0901600), the National Natural Science Foundation of China (Grant Nos. 21603013, 31870055, 31822002), Beijing Natural Science Foundation (8194074), and the Biological Resources Programme (KFJ-BRP-009) from the Chinese Academy of Sciences.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical statement

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

253_2019_10105_MOESM1_ESM.pdf (732 kb)
ESM 1 (PDF 731 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Life Sciences and TechnologyXinjiang UniversityXinjiangPeople’s Republic of China
  2. 2.CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial ResourcesInstitute of Microbiology, Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.University of Chinese Academy of SciencesBeijingPeople’s Republic of China

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