The Establishment And Application Of Pantothenic Acid Synthase As A Target For Anti-tb Drug Screening Model

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC (Rv3602c) gene, catalyzes the essential ATP-dependent condensation of D-pantoate and β-alanine to form pantothenate, and is the key rate-limited enzyme involved in the biosynthesis of pantohenate. Many studies show that pantothenate is essential for the growth of Mycobacterium tuberculosis. Therefore, pantothenate synthetase presents a suitable target for developing anti-tuberculosis drugs.We have cloned the gene panC (Rv3602c) and introduced a site-mutant into it in order to attach a HisTag to C-terminal of the pantothenate synthetase for the convenience of the later protein purification. After successful construction of genetic engineering strain highly expressing recombinant pantothenate synthetase, the expressed products were purified by Ni²⁺ chelating affinity chromatography. In the end, we obtained highly pure recombinant pantothenate synthetase.Based on the enzyme activity assay of pantothenate synthetase, we finally built a high-throughput screening model targeting pantothenate synthetase of Mycobacterium tuberculosis. After screening and rescreening of 2560 kinds of compounds stored in our laboratory, we got six positive compound candidates. Meanwhile, we screened 1227 kinds of microbial fermentations, by comparing with the results of synchronous cell-model screening, 6 compounds and 7 fermentation candidates were acquired.