PATHOBIOCHEMISTRY OF CHLAMYDIA TRACHOMONAS HUMAN SEXUALLY TRANSMITTED DISEASES

E. 大肠杆菌小菌素B17（MccB 17）是一种后修饰的 抑制细菌DNA促旋酶的肽抗生素。 它包含 四个恶唑和四个噻唑环，代表了广泛的 一类药学上重要的天然产物， 元杂环。 仅适用于MccB 17系统有恶唑 和噻唑生物合成酶已被确定，因此， 一种适合于杂环机理表征的体系 阵 MccB 17的产生和活性需要以下物质的存在： 7个质粒编码基因，mcbABCDEFG，除了几个 染色体编码基因 MccB 17的生物合成需要 四个基因的产物，mcbABCD。 mcbA编码无活性的69个氨基酸 酸前体分子preMccB 17 通过McbB的作用， McbC和McbD，preMccB 17经后修饰以产生 proMccB17. 这个过程将四个半胱氨酸和四个丝氨酸 分别为四个噻唑和四个恶唑杂环。 通过染色体编码的26个氨基酸的前导肽去除 蛋白酶导致proMccB 17转化为活性 抗生素MccB 17 操纵子中剩下的三个基因mcbEFG 负责MccB 17的出口和豁免权 我们最近 报道了活性E.大肠杆菌微菌素合成酶，使用 作为一种初级测定，使用识别恶唑的多克隆抗体 和/或噻唑环，以确定McbB，C， 和D是合成酶复合物的成分， 环化、脱水和脱乙酰基的条件和辅因子。 每个酶促翻译后转化的基础是去饱和 甘氨酸-丝氨酸和甘氨酸-半胱氨酸二肽部分转化为氨基-甲基恶唑 和噻唑类抗生素 每次完成的修改结果 在20.03 Da的损失，允许分化的生物合成 通过质谱法（MS）分析中间体。 正在进行的项目 本实验旨在阐明恶唑和噻唑的作用机理 每种酶和先导酶的形成和作用的确立 在这些独特的反应中。 这些研究严重依赖MS 用于生物合成中间体的表征， 所涉及的酶的微观表征。 使用 BUSM设施提供的补充仪器， 分析选定的样本，有关microcin的问题 下面将讨论B17处理。 的到来 在BUSM资源FT-ICR MS将显着扩大能力 特别是对于>5 kDa离子的MS/MS。 1. ATP Stoichiom，~ta. 在我们首先注意到的要求中， 微球蛋白合成酶活性与ATP呈绝对依赖关系。 我们 当前的目标是确定ATP水解的化学计量， 杂环形成。 对于这些实验，反应的等分试样 在对46个氨基酸的底物进行修饰后， 底物和产物将从反应混合物中纯化出来， HPLC，以及起始材料（M）与各种产物的比率 (M-20和M-40）将通过ESIMS进行评估。 将这些数据与 在平行运行的反应中水解的ATP的量将提供 ATP水解与形成的杂环数的比率。 2. 基板处理的整体机制。 对于一种酶来说， 多个类似的修改，一个核心问题是有多少 是否为单个绑定事件完成了修改？ 中 分配机制，答案是一，对于一个过程机制， 对于微菌素，答案将在2和8之间。 初始 在康奈尔大学获得的ESI/FT/FTMS数据有利于分布 机制，但需要进一步的实验。 . 加工方向。 另一个重要的问题是， MS所解决的问题涉及以下物质的方向性或区域选择性： 处理. 换句话说，微菌素合成酶是否会改变其 基质以N-~C、C-~+N或以随机方式。 Tandem MS将 在环化位点的定位中发挥关键作用， 生物合成中间体

E. coli microcin B17 (MccB17) is a posttranslationally modified peptide antibiotic that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five membered heterocycles. Only for the MccB17 system have the oxazole and thiazole biosynthetic enzymes been identified, thus making this system amenable to characterization of the mechanism of heterocycle formation. Production and activity of MccB 17 require the presence of seven plasmid encoded genes, mcbABCDEFG, in addition to several chromosomally encoded genes. Biosynthesis of MccB17 requires the products of four genes, mcbABCD. mcbA codes for an inactive 69 amino acid precursor molecule, preMccB 17. Through the action of McbB, McbC, and McbD, preMccB17 is modified posttranslationally to produce proMccB17. This processing converts four cysteine and four serine residues to four thiazole and four oxazole heterocycles, respectively. Removal of a 26 amino acid leader peptide by a chromosomally encoded protease results in the conversion of proMccB17 to the active antibiotic, MccB17. The remaining three genes in the operon, mcbEFG are responsible for MccB 17 export and immunity. We have recently reported the purification of active E. coli microcin synthetase, using as a primary assay a polyclonal antibody that recognizes the oxazole and/or thiazole rings in modified microcin to establish that McbB, C, and D were constituents of the synthetase complex and to define conditions and cofactors for the cyclization, dehydration and desaturation that underlie each enzymatic posttranslational conversion of gly-ser and gly-cys dipeptide moieties into amino-methyloxazoles and thiazoles in the antibiotic. Each completed modification results in a loss of 20.03 Da, allowing differentiation of biosynthetic intermediates by mass spectrometry (MS). Current projects ongoing in the lab are aimed at elucidating the mechanism of oxazole and thiazole formation and establishing the role of each enzyme and the leader peptide in these unique reactions. These studies rely heavily on MS for the characterization of biosynthetic intermediates andfor the microcharacterization of the enzymes involved. Using the complementary instruments available at the BUSM facility for the analysis of selected samples, the questions about microcin B17processing outlined below will be addressed. The arrival of an FT-ICR MS at the BUSM Resource will markedly expand the capabilities of the lab, particularly for MS/MS of >5 kDa ions. 1. ATP Stoichiom,~ta. Among the requirements that we first noted for microcin synthetase activity was an absolute dependence ox ATP. Our immediate goal is to determine the stoichiometry of ATP hydrolysis to heterocycle formation. For these experiments, aliquots of a reaction following the modification of a 46 amino acid substrate will be taken, the substrate and products will be purified out of the reaction mix by HPLC, and the ratio of starting material (M) to the various products (M-20 and M-40) will be assessed by ESIMS. Comparison of this data to theamount of ATP hydrolyzed in a reaction run in parallel will provide a ratio of ATP hydrolysis to the number of heterocyles formed. 2. Overall Mechanism of Substrate Processing. For an enzyme performing multiple, similar modifications, a central question is how many modifications are completed for a single binding event? In a distributive mechanism, the answer is one, for a processive mechanism, the answer would be between two and eight for microcin. Initial ESI/FT/FTMS data acquired at Cornell University favors a distributive mechanism, although further experiments are required. . Directionali1y of Processing. Another important question that can be addressed by MS concerns the directionality, or regioselectivity, of processing. In other words, does microcin synthetase modify its substrate in a N-~C, C--+N, or in a random fashion. Tandem MS will play a key role in localization of cyclization sites in the biosynthetic intermediates.