当前位置： SCI文献检索 > APPLIED MICROBIOLOGY AND BIOTECHNOLOGY期刊下所有文献 > Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids.

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

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.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Zheng X,Cui Y,Li T,Li R,Guo L,Li D,Wu B

doi

10.1007/s00253-019-10105-9

subject

Has Abstract

pub_date

2019-10-01 00:00:00

pages

8051-8062

issue

eissn

0175-7598

issn

1432-0614

pii

10.1007/s00253-019-10105-9

journal_volume

103

pub_type

杂志文章

在线工具

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