Branched-chain amino acid aminotransferases from Thermus thermophilus and Mycobacterium bovis: Comparative Characterization and Inhibitor Screening

• Main Authors: Pham Minh Tan, 方明堂
• Other Authors: Hwang, Tzann-Shun
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/4h6vs5

碩士 === 中國文化大學 === 生物科技研究所 === 103 === Branched-chain aminotransferases (BCATs) catalyze the transamination of branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine. BCATs have been classified to the fold type IV class of PLP-dependent enzymes. More importantly, the catalysis is on the re-face of the PLP cofactor. It means that BCATs can be used to synthesize L-form of branched-chain amino acids and unnatural amino acids from their respective keto acids; they have broad applicability in the synthesis of fine chemicals and pharmaceuticals. In this study, it first focused on characterization of Thermus thermophilus branched-chain amino acid aminotransferase (TtBCAT). The best assay condition was anticipatedly accomplished for TtBCAT. The kinetic parameters, KM, Vmax and kcat for KIC substrate were 48.1 mM, 63.6 U/mg, 81.6 s-1, respectively and for L-glutamate substrate were 29.6 mM, 64.3 U/mg, 95.3 s-1, respectively. MbBCAT was reported to be the key enzyme for methionine synthesis in M. bovis. Comparison between TtBCAT and MbBCAT was conducted, and it was found that TtBCAT was very different from MbBCAT in sequence, molecular weight, metal effects, DTT effects and substrate specificity. Blocking of methionine synthesis in M. bovis was a considerable solution to inhibit the growth of M. bovis; therefore, screening inhibitors was a good way to develop medicine against MbBCAT. In order to construct an active form of MbBCAT for an effective docking by in silico screening. PMP cofactor of MbBCAT was replaced by PLP cofactor, and then, PLP-form – MbBCAT was used as target for autodocking software perform in silico screening of approximately 100,000 compounds. 10 compounds with the highest affinity to MbBCAT were chosen. Evaluating experiments were carried out to determine inhibitory effect on MbBCAT. Absolutely, all of those candidates inhibited MbBCAT enzyme, nonetheless compound 4 was found to have the best inhibitory effect with IC50 and Ki values of 2.37 and 0.45 μM, respectively. This candidate was potential in developing drug against Mycobacterium bovis because it was beneficial in the structure-based chemical design.