Investigation of the structure and function of urea amidolyase

尿素酰胺裂解酶的结构和功能研究

基本信息

批准号：
8367184
负责人：
Martin Raoul St. Maurice
金额：
$ 34.62万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8367184
关键词：
Active Sites Algae Amidohydrolases Amino Acids Ammonia Bacteria Binding Biotin Biotin carboxylase Candida Candida albicans Carbon Dioxide Catalysis Cloning Complement Crystallization Crystallography Disseminated candidiasis Enzyme Inhibitor Drugs Enzyme Inhibitors Enzyme Kinetics Enzymes Family Future Genetic Screening Goals Growth Human Hydrolase Hydrolysis Hyphae Individual Infection Investigation Kinetics Maps Mediating Methods Modeling Molecular Molecular Structure Multienzyme Complexes Mutagenesis Mutation Organism Pathogenicity Plasmids Prokaryotic Cells Protein Engineering Proteins Pyruvate Carboxylase Recombinants Research Resolution Roentgen Rays Role Saccharomyces cerevisiae Series Site Site-Directed Mutagenesis Structure Systemic infection Therapeutic Urea X-Ray Crystallography Yeasts allophanate amidase analog combat design enzyme activity enzyme structure expression vector family structure fungus grasp inhibitor/antagonist insight knockout gene macrophage member novel pathogen research study tool yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Urea amidolyase (UAL) is a multi-functional, biotin-dependent enzyme that catalyzes the ATP-dependent hydrolysis of urea to ammonia and carbon dioxide in yeast, algae and prokaryotes. The enzyme activity influences a yeast-to-hyphae switch that allows Candida albicans to escape from macrophages during systemic infections. Unlike other members of the biotin-dependent enzyme family, the structure and function of UAL has not been well characterized. This project will use protein X-ray crystallography to determine the molecular structure of the enzyme active sites of UAL and will combine crystallography with protein engineering and steady-state kinetics to clarify the mechanism of catalysis and intermediate channeling in UAL. The subunit composition and oligomerization state of UAL makes this enzyme particularly amenable to structural studies and as a model for biotin-dependent enzyme catalysis. The kinetic analysis of site-specific mutations and domain truncations will provide valuable insights into the mechanism of catalysis in UAL and will further the description of catalysis in the important biotin-dependent enzyme family. Both Saccharomyces cerevisiae and C. albicans require UAL for urea-dependent growth. This requirement will be exploited to establish a genetic screen in S. cerevisiae that will serve as a powerful tool to probe functional regions and residues in UAL and which will broadly impact the description of enzyme structure and function in all biotin-dependent enzymes. PUBLIC HEALTH RELEVANCE: Candida albicans is the most important and common fungal pathogen in humans. The enzyme urea amidolyase (UAL) contributes to pathogenicity by initiating a morphological switch that permits the organism to transition from unicellular yeasts t multicellular filamentous hyphae. Since UAL is found only in a subset of bacteria and fungi, it represents a promising and specific target to combat Candida infections. This project will characterize the structure and function of urea amidolyase using X-ray crystallography, mutagenesis and enzyme kinetics. Achieving the goals of this project will result in a greatly enhanced description of UAL structure and function, providing necessary insights for the eventual design of UAL-specific inhibitors with therapeutic potential.

描述（由申请人提供）：尿素酰化酶（UAL）是一种多功能的生物素依赖性酶，可在酵母，藻类和原核中催化尿素与氨和二氧化碳的ATP依赖性水解。酶活性会影响酵母到河口开关，该开关使白色念珠菌在全身感染过程中逃脱了巨噬细胞。与依赖生物素依赖性酶家族的其他成员不同，UAL的结构和功能尚未得到很好的特征。该项目将使用蛋白质X射线晶体学来确定UAL酶活性位点的分子结构，并将晶体学与蛋白质工程和稳态动力学结合，以阐明UAL中催化和中间通道的机制。 UAL的亚基组成和寡聚状态使该酶特别适合结构研究，并作为生物素依赖性酶催化的模型。位点特异性突变和结构截面的动力学分析将为UAL中催化机理提供宝贵的见解，并将进一步描述重要的生物素依赖性酶家族中的催化。酿酒酵母和白色念珠菌都需要尿素依赖性生长。该要求将被利用以在酿酒酵母中建立遗传筛查，该遗传筛选将作为探测UAL中功能区域和残基的强大工具，并将广泛影响所有依赖生物素依赖性酶的酶结构和功能的描述。公共卫生相关性：白色念珠菌是人类中最重要，最常见的真菌病原体。酶尿素酰化酶（UAL）通过启动形态学转换允许生物体从单细胞酵母t多细胞丝状丝菌过渡来促进致病性。由于仅在细菌和真菌的一部分中发现了UAL，因此它代表了对抗念珠菌感染的有前途和特定的靶标。该项目将使用X射线晶体学，诱变和酶动力学来表征尿素酰化酶的结构和功能。实现该项目的目标将极大地增强对UAL结构和功能的描述，为最终设计具有治疗潜力的UAL特异性抑制剂提供必要的见解。