台勾霉素B生物合成通路中两个后修饰酶TiaS5和TiaM的催化机制研究

Tiacumicin B is a narrow-spectrum antibiotic that shows activities against various Gram-positive pathogenic bacteria and breast cancer cells. In 2011, Tiacumicin B was approved for the treatment of Clostridium difficile infection (CDI), which has become an important disease among hospitalizations. Methylation and halogenation in Tiacumicin B biosynthesis, catalyzed by TiaS5 and TiaM, evidently improve the activity of Tiacumicin B. By now, the catalytic mechanisms of TiaS5 and TiaM are unclear. In this project, we will investigate the catalytic mechanisms of TiaS5 and TiaM using x-ray crystallography. We aim to determine the apo and complex structure of TiaS5 and TiaM. We will also carry out the enzyme kinetics experiments for wild type and mutants of TiaS5 and TiaM. Currently, we have obtained large amount of homogeneous TiaS5 and TiaM. At the same time, we have collected a dataset at 3.7 Å and obtained a preliminary Molecular Replacement solution for TiaS5.

台勾霉素B是一种具有革兰氏阳性菌抗性和抗乳腺癌细胞活性的窄谱抗生素，已于2011年被FDA批准用于艰难梭菌感染（CDI）的治疗。TiaS5和TiaM是台勾霉素B生物合成过程中的两个后修饰酶，其催化的甲基化和卤素化修饰对台勾霉素B的抗菌活性有显著影响。目前，这两个酶的催化机理尚不清楚。本项目拟应用结构生物学方法来研究TiaS5和TiaM的催化机理。通过解析酶及其与底物复合物的高分辨率晶体结构，结合野生型和关键位点突变体的酶动力学实验来研究其催化机制和底物特异性的分子基础，为台勾霉素B生物合成通路研究和更高效地进行台勾霉素B的生物合成提供理论支持。目前，我们已获得TiaS5和TiaM纯度高、均一性好的蛋白，并且已经收了一套TiaS 5的3.7Å的数据，初步确定了其分子置换解。