嗜水气单胞菌CphA酶与碳青霉烯类抗生素之间的分子识别和相互作用

发布时间：2018-05-06 04:12

本文选题：CphA酶 + 亚胺培南　；参考：《西北大学》2017年硕士论文

【摘要】：金属β-内酰胺酶(Metallo-beta lactamase,MβLs)通过水解β-内酰胺抗生素的β-内酰胺环而阻止抗生素对抗细菌感染的有益行为。来自于嗜水气单胞菌的CphA酶是MβLs亚群的一员,其在活性位点只有一个Zn2+,并只特异性水解碳青霉烯类抗生素。然而对于CphA酶与碳青霉烯类抗生素之间的分子识别和相互作用知之甚少。本研究将在重组菌中表达并分离纯化得到CphA酶的基础上,采用荧光光谱法和分子对接模拟法相结合,以亚胺培南(imipenem,IMP)和比阿培南(biapenem,BIA)为研究对象,研究它们在结合过程中,酶的构象变化、抗生素分子的空间分布取向、形成络合物的本质以及它们之间的作用力类型和作用位点数等。(1)将含有pET28b-CphA的大肠杆菌BL21(DE3)进行诱导表达,通过阳离子交换层析分离纯化,然后用SDS-PAGE鉴定和Bradford测蛋白含量,最后对其进行荧光表征。结果表明:得到有活性的纯度较高的样品,其分子量约为28 KDa,纯化后产量约为0.18～0.21 mg/mL,当激发波长为278 nm时,发现在336.2 nm处荧光明显。(2)在 277 K、pH 7.4 PBS 中,测得 CphA 酶对 IMP 与 BIA 的 Km 分别为 356μM和286 μM;通过荧光发射光谱测得CphA酶与IMP和BIA的分子饱和比分别为0.95和0.98;同步荧光光谱结果表明:它们相互作用且Tyr残基附近的微环境发生微弱的变化;测定277 K、281 K和285 K三个温度下CphA酶分别对IMP和BIA结合过程的猝灭光谱,结果表明:它们之间发生的猝灭均为静态猝灭;它们之间是依靠单一的结合位点结合,且均为自发的放热过程。(3)分子对接结果显示CphA酶与IMP和BIA在结合过程中存在一些相同之处:两种抗生素β-内酰胺环的C3羧基氧直接与Zn2+形成金属配位键,促进相互作用,且均使CphA酶loop环发生局部的构象变化。不同之处为:IMP因侧链空间位阻较小,使其全部进入口袋,在相互作用过程中,共有3组静电力和5组氢键形成;BIA因侧链双环三唑的位阻比较大,留部分侧链位于口袋外侧,在相互作用过程中,共有4组静电力和2组氢键,进而使得CphA酶对BIA的亲和力大于对IMP的,以及形成CphA-BIA复合物的稳定性也大于CphA-IMP复合物的稳定性。荧光光谱结果中CphA-BIA复合物体系的Ksv和Ka高于CphA-IMP复合物体系的,也证实这一推断。另外,对接结果显示复合物体系△G为负值,表明两个复合物的形成均属于自发的放热过程,与前期热力学结果一致。
[Abstract]:Metal beta lactamases (Metallo-beta lactamase, M beta Ls) inhibit the beneficial behavior of antibiotics against bacterial infection by hydrolysis of beta lactam beta lactam rings. The CphA enzyme from Aeromonas hydrophila is a member of the M beta Ls subgroup, with only one Zn2+ at the active site and only specifically hydrolyzed carbapenems. There is little knowledge about the molecular recognition and interaction between CphA enzyme and carbapenems. On the basis of the expression and separation and purification of CphA enzymes in the recombinant bacteria, the study is based on the combination of fluorescence spectroscopy and molecular docking simulation, and the research object is imipenem (imipenem, IMP) and apenem (biapenem, BIA). The conformation changes of the enzyme, the spatial distribution orientation of the antibiotic molecules, the nature of the complex and the number of action points between them, and so on. (1) the BL21 (DE3) containing pET28b-CphA was induced and expressed by the cation exchange chromatography, and then identified by SDS-PAGE and B The protein content was measured by Radford. The results showed that the high purity samples were obtained with a molecular weight of about 28 KDa and the yield was about 0.18 ~ 0.21 mg/mL after purification. When the excitation wavelength was 278 nm, the fluorescence was found at 336.2 nm. (2) the IMP and BIA Km were measured in 277 K and pH 7.4 PBS. The molecular saturation ratio of CphA enzyme to IMP and BIA was 0.95 and 0.98, respectively, by fluorescence emission spectra, respectively. The results of synchronous fluorescence spectra showed that they interact with the microenvironment of Tyr residues in the microenvironment slightly, and the CphA enzyme of 277 K, 281 K and 285 K three temperatures was sudden, and the CphA enzyme in the binding process of IMP and BIA, respectively. The quenching spectrum shows that the quenching between them is static quenching; they are dependent on a single binding site and are spontaneous exothermic processes. (3) molecular docking results show that there are some similarities between CphA enzyme and IMP and BIA in the process of binding: the C3 carboxyl oxygen of the two antibiotics beta lactam ring is directly and Zn2+ shaped. Metal coordination bonds promote interaction and make local conformation changes of the CphA loop ring. The difference is that IMP has 3 groups of static electricity and 5 groups of hydrogen bonds in the process of interaction, because the side chain space has smaller steric resistance, and the side chain of the side chain of double ring three azole is relatively large, and the remaining side chain is located outside the pocket. Side, in the process of interaction, there are 4 groups of static electricity and 2 groups of hydrogen bonds, which make the affinity of CphA enzyme to BIA greater than that of IMP, and the stability of the CphA-BIA complex is greater than the stability of CphA-IMP complex. The Ksv and Ka of the CphA-BIA complex system in the fluorescence spectrum is higher than the CphA-IMP complex system, and this also confirms this In addition, the docking results show that the complex system Delta G is negative, indicating that the formation of the two complexes all belong to the spontaneous exothermic process, which is in accordance with the previous thermodynamic results.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】