title: Identification of novel thermostable taurine-pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis.
abstract: ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine-pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTAgth) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its K M and v max values for taurine were 5.3 mM and 0.28 μmol s(-1) mg(-1), respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTAgth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTAgth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTAgth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, People's Republic of China.
authors.forename: Yujie
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, People's Republic of China. charlesdyi@yahoo.com.
authors.forename: Dong
authors.identifier: http://orcid.org/0000-0003-2594-0464
authors.initials: D
authors.lastname: Yi
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, People's Republic of China.
authors.forename: Shuiqin
authors.initials: S
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, 200237, Shanghai, People's Republic of China. dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000588:Amines / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056506:Geobacillus / Q000737:chemistry* / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D062105:Molecular Docking Simulation; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013654:Taurine / Q000737:chemistry* / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000588:Amines; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013654:Taurine; D011733:Pyridoxamine; D019289:Pyruvic Acid; D000637:Transaminases; C010627:pyridoxamine phosphate
keywords: Biocatalysis; Chiral amine; Taurine–pyruvate transaminase; Thermostable transaminase; ω-Transaminase