金黄色葡萄球菌一氧化氮自由基诱导乳酸脱氢酶晶体结构解析和酶活性研究

发布时间：2018-06-25 09:20

本文选题：金黄色葡萄球菌 + 一氧化氮自由基　；参考：《天津医科大学》2009年博士论文

【摘要】： 研究背景：金黄色葡萄球菌(Staphylococcus aureus)是最常见的革兰阳性致病菌，可以引起人体种类众多的感染。而且，随着抗生素的广泛使用，耐药性问题越来越严重。万古霉素作为治疗耐药金黄色葡萄球菌感染的最后一道防线，也出现了敏感性降低，甚至完全耐药的现象。其实，人类的自身先天免疫系统是有效抗击外来微生物入侵的第一道防线。其中一种重要的机制就是诱导性一氧化氮合成酶系统，其所合成的NO~*可以直接抑制微生物的有氧呼吸能量代谢，并抑制微生物的DNA复制。而金黄色葡萄球菌区别于其他非致病性葡萄球菌的一个主要特征，就是可以抵抗NO~*的毒性，在外源性NO~*压力下仍能生长和繁殖。其NO~*解毒机制一方面与黄素血红蛋白(hmp SACOL0220)行使的NO~*双氧化酶功能有关，可以将毒性NO~*转化为无毒的硝酸；另一方面与hmp同处于一个操纵子中的金黄色葡萄球菌乳酸脱氢酶-1(ldh-1 SACOL022)的诱导高表达密切相关。ldh-1与金黄色葡萄球菌中持续表达的同功酶ldh-2可以改变金黄色葡萄球菌在NO~*生存环境下细菌的能量代谢途径，从而使金黄色葡萄球菌可以在NO~*环境压力下正常生长和繁殖。研究思路：理论上ldh-1蛋白可以作为一个抗金黄色葡萄球菌的潜在靶点，任何对ldh-1具有选择性抑制作用的小分子物质，即只抑制金黄色葡萄球菌ldh-1但对人类的乳酸脱氢酶同功酶无明显抑制作用，就可能成为一种新的抗生素。研究方法：本研究首先将金黄色葡萄球菌中编码ldh-1和ldh-2的基因全长克隆到表达载体pET-28a中，大量表达重组蛋白，再利用重组蛋白的Histag，以亲和层析法和分子筛两步联合方法纯化重组蛋白纯度超过95％。进而，，在不同理化条件下促使蛋白结晶，然后用所得到的单晶进行X射线衍射以解析蛋白的三维机构。研究结果：l；dh-1蛋白结晶，并收集高质量衍射数据。初步分析属于C2空间群，晶胞参数为a=131．36，b=74．362，c=103．243。ldh-1的结构最终可以被精修到2．2 (?)，R-Factor为17％，Rfree-factor为23．4％。使用分子置换法解析出其三维结构，分辨率达2．2埃。每个晶胞中有两个不对称的乳酸脱氢酶分子。每个ldh-1分子中包含两个结构域。大的结构域由22-160和248-264号氨基酸形成具有典型Rossmann特征的折叠。通过蛋白一级结构氨基酸序列的多重序列比对，活性位点的关键氨基酸序列保守，但活性环区上翘，使活性位点更加暴露。本研究还对ldh-1、ldh-2、人类骨骼肌ldh-A和心肌ldh-B的酶催化活性进行了比较，发现ldh-1与底物和辅酶的亲和性，和反应效率均明显高于人类的同功酶。通过对金黄色葡萄球菌中1dh结构、功能的研究，希望能以酶结构为基础，使用工作站筛选相关类别的小分子数据库，发现潜在的结构抑制剂。
[Abstract]:Background: Staphylococcus aureus is the most common gram-positive bacteria, which can cause a variety of human infections. Moreover, with the widespread use of antibiotics, the problem of drug resistance is becoming more and more serious. Vancomycin, as the last line of defense in the treatment of drug-resistant Staphylococcus aureus infection, also has the phenomenon of reduced sensitivity and even complete drug resistance. In fact, the human innate immune system is the first line of defense against the invasion of foreign microorganisms. One of the important mechanisms is the inducible nitric oxide synthase system, which can directly inhibit the aerobic respiration energy metabolism of microorganisms and inhibit the DNA replication of microbes. One of the main characteristics of Staphylococcus aureus differs from other non-pathogenic staphylococci is that it can resist the toxicity of NOA * and can still grow and reproduce under the pressure of exogenous NOA *. The detoxification mechanism is related to the function of hmp SACOL0220, which can transform noxotoxin into nontoxic nitric acid. On the other hand, the induced overexpression of staphylococcus aureus lactate dehydrogenase 1 (ldh-1 SACOL022) in the same operon with hmp was closely related to the sustained expression of isoenzyme ldh-2 in Staphylococcus aureus, which could change the expression of Staphylococcus aureus. The energy metabolism pathway of bacteria in the living environment, Thus Staphylococcus aureus can grow and reproduce normally under environmental pressure. Research idea: theoretically, ldh-1 protein can be used as a potential target against Staphylococcus aureus, any small molecule with selective inhibitory effect on ldh-1. It may become a new antibiotic to inhibit only Staphylococcus aureus ldh-1 but no obvious inhibition of human lactate dehydrogenase isoenzyme. Methods: in this study, the full-length gene encoding ldh-1 and ldh-2 in Staphylococcus aureus was cloned into the expression vector pET-28a to express a large number of recombinant proteins. The purity of recombinant protein was more than 95% by affinity chromatography and molecular sieve. Furthermore, the protein was crystallized under different physicochemical conditions, and then X-ray diffraction was carried out with the obtained single crystal to analyze the three-dimensional mechanism of the protein. Results the protein was crystallized and high quality diffraction data were collected. The preliminary analysis belongs to the C2 space group, and the unit cell parameter is a 131.36 / 74.362C ~ (-1) 103.243.ldh-1 structure which can be refined to 2.2 (?) R-factor 17 ~ (th) Rfree-factor = 23.4 ~ (th) ~ (th). The three-dimensional structure was analyzed by molecular replacement method with a resolution of 2.2 A. There are two asymmetric lactate dehydrogenase molecules in each unit cell. Each ldh-1 molecule contains two domains. The large domain consists of 22-160 and 248-264 amino acids to form folding with typical Rossmann characteristics. The key amino acid sequence of the active site is conserved by multiple sequence alignment of amino acid sequence of the primary structure of protein, but the active ring region is upwarped, which makes the active site more exposed. The enzyme catalytic activity of ldh-1 ldh-2, human skeletal muscle ldh-A and myocardial ldh-B was compared. It was found that the affinity and reaction efficiency of ldh-1 to substrate and coenzyme were significantly higher than that of human isozyme. Through the study of the structure and function of 1dh in Staphylococcus aureus, it is hoped that based on the enzyme structure, a workstation can be used to screen the relevant small molecular databases and to find potential structural inhibitors.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378;R515

【参考文献】