茶叶提取物中1-脱氧-D-木酮糖5-磷酸还原异构化酶抑制剂的筛选及茶叶抗菌机制的研究

1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the first rate limiting enzyme in the non-mevalonate pathway (MEP) pathway for terpenoid biosynthesis, is an attractive target for seeking novel antimicrobial agents. A lot of research has shown that tea extracts possess antimicrobial activity. Through previous studies, we found that tea extracts showed quite strong DXR inhibitory effect in vitro and the activity obviously concentrated in their ethyl acetate and n-butanol extracts, respectively. After further analysis of the ethyl acetate parts, we realized that the major active compounds were polyphenols. Based on these, the inhibitory specificity and the inhibition kinetics of the compounds against DXR were investigated and the interactions between DXR and these polyphenols were simulated through molecular docking. In this project, we plan to analyze the DXR inhibitory activity of the n-butanol parts of tea extracts, expecting to obtain better DXR inhibitors and then elucidate their inhibitory mechanisms against DXR. More important, we plan to explore whether these DXR inhibitors isolated from the tea extracts can suppress the activity of DXR in vivo, blocking the MEP terpenoid biosynthetic pathway of bacteria, and thus acting as antimicrobial agents. And on this basis, we would try to further deduce the antibacterial mechanism of tea.

1-脱氧-D-木酮糖-5-磷酸还原异构化酶（DXR）是细菌非甲羟戊酸（MEP）途径中的关键限速酶，也是筛选新型抗菌化合物的有效靶标。研究表明，茶叶提取物具有抗菌活性。本课题前期研究发现茶叶提取物在体外有较好的DXR抑制活性，并且该活性明显集中在提取物的乙酸乙酯和正丁醇萃取部分。在对乙酸乙酯萃取部分进行分析后发现其中几种茶多酚化合物显示出较好的DXR抑制活性。在此基础上我们深入探讨了这些化合物对DXR抑制的特异性及抑制动力学，并利用分子对接技术模拟了它们与DXR之间的相互作用。本项目拟进一步分析正丁醇萃取部分的DXR抑制活性，以期从中分离获得更好的DXR抑制剂并揭示它们抑制DXR的机制。更重要的是，本项目还拟在细胞水平上探讨茶叶提取物中的这些DXR抑制剂是否可以通过抑制细菌体内DXR的活性，从而阻断细菌的萜类化合物MEP生物合成途径，进而起到抗菌效果，并在此基础上进一步阐明茶叶抗菌机制。

DXR 作为 MEP 途径上的关键限速酶，是细菌体内合成萜类化合物的一个关键节点，是筛选新型抗菌化合物的有效靶标。前期研究发现茶叶提取物在体外有较好的DXR抑制活性，并且该活性明显集中在提取物的乙酸乙酯和正丁醇萃取部分。在对乙酸乙酯萃取部分进行分析后发现其中几种儿茶素、茶黄素化合物显示出较好的DXR抑制活性。在此基础上本项目通过多种分离手段分离并跟踪茶叶提取物正丁醇萃取部分的DXR抑制活性，发现花青素类化合物原花青素C1、原花青素 B2、肉桂单宁A2、肉桂单宁D1、肉桂单宁B1有DXR蛋白抑制活性，随后实验用双倒数曲线法测定发现这些筛选化合物相对底物DXP是非竞争性抑制，相对于底物NADPH是反竞争性抑制；通过动态光散射扫描等方法证实这些化合物对DXR是一种特异性抑制。. 前期研究也表明，抑制细菌 DXR 酶很可能是茶多酚及茶黄素化合物一个重要抗菌机理，但要证实茶多酚及茶黄素等化合物的这一抗菌机制，还需要细菌水平上研究这些化合物是否能够通过抑制菌体中 DXR 酶的活性。本项目通过实验证实：①将大肠杆菌改造成能够过表达DXR蛋白的菌种，能显著逆转儿茶素、茶黄素化合物对大肠杆菌菌体的生长抑制；②当细菌培养液中加入儿茶素、茶黄素化合物能显著影响工程菌对萜类化合物番茄红素的表达。这些研究结果支持抑制细菌DXR蛋白极可能是茶叶提取物一个重要抗菌机理。