Terminal enzymes of heme synthesis

描述(由申请人提供)：拟议工作的总体目标是获得数据，以便在分子水平上描述末端的两种血红素合成途径酶，原卟啉原氧化酶(PRO)和铁络合酶是如何发挥作用的。本提案的目标是确定人PRO和铁络合酶以及细菌氧非依赖性PRO的催化和结构特征。潜在的蛋白质-蛋白质相互作用涉及这些酶以及细胞色素c和Frataxin将被研究。获得的数据将描述人类自然发生的突变导致遗传性疾病--卟啉症--的催化后果，这在患者治疗和咨询方面应该是有价值的。这些数据还将识别和描述人类和微生物酶之间存在的任何关键差异。这些结果可能为新型抗菌剂的开发奠定基础。这项建议的具体目的是：1.)通过动力学、定点突变和基于结构的研究，确定人PRO的结构-功能特征，确定细胞色素c是否是PPO的真正电子受体，并表征这种相互作用，并分离和鉴定细菌的氧非依赖性原卟啉原氧化酶。2.)通过使用动力学、定点突变和基于结构的研究来定义铁络合酶的结构-功能特征，表征铁络合酶：Frataxin的相互作用并确定其与环状铁母细胞ERR患者的相关性，以及通过定点突变和体检来表征动物和细菌铁络合酶中的[2Fe-2S]簇。3.)鉴定和表征铁络合酶和多酚氧化酶之间的蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): The overall goals of the proposed work are to obtain data that will allow descriptions at the molecular level how the terminal two heme synthetic pathway enzymes, protoporphyrinogen oxidase (PRO) and ferrochelatase, function. The goals of the current proposal are to define catalytic and structural features of human PRO and ferrochelatase and bacterial oxygen-independent PRO. Potential protein-protein interactions involving these enzymes as well as cytochrome c and frataxin will be examined. The data obtained will describe the catalytic consequences of naturally occurring mutations in humans that lead to the inherited diseases known as porphyrias, and this should be of value in patient treatment and counseling. The data will also identify and characterize any key differences that exist between the human and microbial enzymes. These results may set the stage for development of new classes of antimicrobial agents. Specific aims of this proposal are to: 1.) define structure-function characteristics of human PRO through the use of kinetics, site-directed mutagenesis, and structure-based studies, determine if cytochrome c is a bona fide electron acceptor for PPO and characterize this interaction, and isolate and characterize bacterial oxygen-independent protoporphyrinogen oxidase. 2.) define structure-function characteristics of ferrochelatase through the use of kinetics, site-directed mutagenesis, and structure-based studies, characterize the ferrochelatase:frataxin interaction and determine its relevance to ERR patients with ringed sideroblasts, and characterize the [2Fe-2S] cluster in animal and bacterial ferrochelatases through site-directed mutagenesis and physical examination. 3.) identify and characterize protein-protein interactions between ferrochelatase and PPO.