Structure Function Analysis of TB Pyrazinamidase

描述(申请人摘要)：吡嗪酰胺(PZA)是一种重要的 目前的短程结核病(TB)化疗的组成部分。PZA游戏 在将治疗缩短到6个月方面发挥了独特的作用，因为它杀死了 未被其他结核病杀死的半休眠结核杆菌种群 毒品。PZA是一种需要转化为活性形式的吡嗪酸的前药 通过细菌烟酰胺酶/吡氨酰胺酶(PZase)杀灭结核分枝杆菌。我们 已经确定了烟酰胺酶/PZase基因(PncA)，并表明突变 在PNcA中，影响PZA活性的因素会导致结核杆菌对PZA产生抗性。 从最近的研究中发现了一个有趣的现象：pncA基因突变 在对PZA耐药的临床分离株中，分离株高度多样化，分布在 PncA基因。很难将pncA突变和pncA水平联系起来 PZase活性与抗性水平有关。我们假设并不是所有的pnca 突变在影响PZase活性和水平方面同样重要 PZA抵抗。在此建议中，我们建议使用 分子遗传学、生化和结构方法的结合。这个 该项目的具体目标是：(1)表征pncA突变在 耐PZA的结核分枝杆菌及其与血清PZA水平的相关性 PZase活性和对PZA的抗性。耐PZA的结核分枝杆菌临床分离株将 分析pncA突变及其与血浆中 用更灵敏的Cl4-PZA PZase测定PZase活性和抗性水平 药敏试验和PZA药敏试验方法。(2)确定活动地点 点突变检测pncA酶的残留量。催化位残留物 将通过对突变体进行基于PCR的定点突变来鉴定 改变所需氨基酸残基的寡核苷酸引物。突变型 PZase将在大肠杆菌中高效表达并纯化，用于PZase的检测。(3)至 对吡嗪酰胺酶(PZase)和 野生型和突变型pncA酶的烟酰胺酶(NAMase)活性。酶 纯化的野生型和突变型pncA酶的动力学将被确定 并进行了比较。亚铁离子可能提高酶活性的假说 副产品的去除将进行测试。(4)测定化合物的晶体结构 野生型和突变型PncA酶。任何pncA酶的晶体结构 是未知的。野生型和突变型pncA酶的结构将是 决心理解PncA基因突变的多样性 引起PZA抵抗。这些研究将提供有关 PZA的作用和抵抗机制，并可能有助于设计新的 抗结核药物。

DESCRIPTION (the Applicant's Abstract): Pyrazinamide (PZA) is an important component of the current short course tuberculosis (TB) chemotherapy. PZA plays a unique role in shortening the therapy to 6 months because it kills a population of semi-dormant tubercie bacilli that are not killed by other TB drugs. PZA is a prodrug that requires conversion to active form pyrazinoic acid by bacterial nicotinamidase/pyrazinamidase (PZase) to kill M. tuberculosis. We have identified the nicotinamidase/ PZase gene (pncA) and shown that mutations in pncA that affect PZA activation cause PZA resistance in tubercie bacillus. An intriguing observation from recent studies is that pncA mutations identified in PZA-resistant clinical isolates are highly diverse and scattered along the pncA gene. It has been difficult to correlate pncA mutations and the level of PZase activity with the level of resistance. We hypothesize that not all pncA mutations are equally important in affecting PZase activity and the level of PZA resistance. In this proposal, we propose to address this hypothesis using a combination of molecular genetic, biochemical and structural approaches. The specific aims of the project are: (1) To characterize pncA mutations in PZA-resistant M. tuberculosis and assess their correlation with the level of PZase activity and PZA resistance. PZA-resistant clinical isolates of MTB will be analyzed in terms of pncA mutations and their correlation with the level of PZase activity and the level of resistance using more sensitive Cl 4-PZA PZase assay and PZA susceptibility testing method. (2) To identify the active site residues of PncA enzyme by site-directed mutagenesis. Catalytic site residues will be identified using PCR based site-directed mutagenesis with mutant oligonucleotide primers that alter desired amino acid residues. The mutant type PZase will be over expressed in E. coli and purified for PZase assay. (3) To carry out a full kinetic analysis of the pyrazinamidase (PZase) and nicotinamidase (NAMase) activities of wild type and mutant PncA enzymes. Enzyme kinetics for both purified wild type and mutant PncA enzymes will be determined and compared. The hypothesis that ferrous ion might enhance the enzyme activity by product removal will be tested. (4) To determine the crystal structure of wild type and mutant type PncA enzymes. Crystal structure of any PncA enzymes is unknown. The structure of wild type and mutant PncA enzymes will be determined to understand the paradox of how very diverse pncA mutations can all cause PZA resistance. These studies will provide important information about mechanisms of PZA action and resistance and may help design new antituberculosis drugs.