Structure Function Analysis of TB Pyrazinamidase

吡嗪酰胺（PZA）是一种重要的抗氧化剂。 目前短程结核病（TB）化疗的组成部分。PZA戏剧 将治疗时间缩短到6个月的独特作用， 未被其他结核病杀死的半休眠结核杆菌群 毒品PZA是一种前药，需要转化为活性形式吡嗪酸 通过细菌烟酰胺酶/吡嗪酰胺酶（PZase）杀死M.结核我们 已经鉴定了烟酰胺酶/ PZase基因（pncA），并表明突变 在pncA中，影响PZA活化导致结核杆菌中的PZA抗性。 最近研究中一个有趣的观察结果是， 在PZA耐药的临床分离株是高度多样化的，分散在沿着， pncA基因。很难将pncA突变与肿瘤的水平相关联。 PZase活性与抗性水平有关。我们假设不是所有的pncA 突变在影响PZase活性和蛋白水平方面同样重要。 PZA抵抗。在本提案中，我们建议使用 分子遗传学、生物化学和结构方法的组合。的 该项目的具体目标是：（1）表征pncA突变， 抗PZA M.结核病，并评估其与水平的相关性 PZase活性和PZA抗性。结核分枝杆菌的PZA耐药临床分离株将 根据pncA突变及其与肿瘤水平的相关性进行分析。 PZase活性和使用更敏感的Cl 4-PZA PZase的抗性水平 测定和PZA敏感性测试方法。(2)要识别活性位点， PncA酶的残基通过定点诱变。催化位点残基 将使用基于PCR的定点突变进行鉴定， 改变所需氨基酸残基的寡核苷酸引物。突变型 PZase在E. coli中，并纯化用于PZase测定。(3)到 对吡嗪酰胺酶（PZase）进行全面的动力学分析， 野生型和突变体PncA酶的烟酰胺酶（NAMase）活性。酶 将测定纯化的野生型和突变体PncA酶的动力学 和比较亚铁离子可能增强酶活性的假说 将测试副产物去除。(4)为了确定晶体结构 野生型和突变型PncA酶。任何PncA酶的晶体结构 不明野生型和突变体PncA酶的结构将被描述为： 下定决心要了解非常多样的pncA突变是如何 导致PZA耐药。这些研究将提供重要信息， PZA的作用和耐药性的机制，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.