Investigation of the structure and function of urea amidolyase

描述(申请人提供)：尿素氨解酶(UAL)是一种多功能的、依赖生物素的酶，在酵母、藻类和原核生物中催化依赖于ATP的尿素水解氨和二氧化碳。这种酶的活性会影响酵母到菌丝的转换，使白色念珠菌在全身感染期间从巨噬细胞中逃脱。与生物素依赖酶家族的其他成员不同，UAL的结构和功能尚未得到很好的描述。该项目将使用蛋白质X射线结晶学来确定UAL酶活性部位的分子结构，并将结晶学与蛋白质工程和稳态动力学相结合，以阐明UAL中的催化和中间通道的机制。UAL的亚基组成和寡聚化状态使其特别适合于结构研究，并作为生物素依赖的酶催化的模型。定点突变和结构域截断的动力学分析将为了解UAL的催化机制提供有价值的见解，并将进一步描述重要的生物素依赖酶家族的催化作用。酿酒酵母和白色念珠菌都需要UAL来实现尿素依赖型生长。这一要求将被用来在酿酒酵母中建立遗传筛查，这将成为探索UAL中功能区和残基的强大工具，并将广泛影响对所有生物素依赖酶的酶结构和功能的描述。 公共卫生相关性：白色念珠菌是人类最重要和最常见的真菌病原体。尿素氨解酶(UAL)通过启动单细胞酵母菌向多细胞丝状菌丝状菌丝转化的形态转换来促进致病性。由于UAL只在一小部分细菌和真菌中发现，它代表了对抗念珠菌感染的一个有前途的特定靶点。本项目将利用X射线结晶学、诱变和酶动力学来表征尿素酰胺酶的结构和功能。实现该项目的目标将大大加强对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。

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.