Investigation of the structure and function of urea amidolyase

描述（由申请人提供）：尿素酰胺解酶（UAL）是一种多功能生物素依赖性酶，在酵母、藻类和原核生物中催化尿素的ATP依赖性水解为氨和二氧化碳。这种酶的活性影响酵母菌到菌丝的转换，使白色念珠菌在全身感染期间从巨噬细胞中逃逸。与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表征。本项目将利用蛋白质X射线晶体学确定UAL酶活性位点的分子结构，并将联合收割机晶体学与蛋白质工程和稳态动力学相结合，阐明UAL中的催化和中间通道机制。UAL的亚基组成和寡聚化状态使得这种酶特别适合于结构研究，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域截短的动力学分析将提供有价值的见解，在UAL的催化机制，并将进一步在重要的生物素依赖性酶家族的催化的描述。酿酒酵母和C.白色念珠菌需要UAL用于尿素依赖性生长。这一要求将被用来建立一个遗传筛选的S。这将作为一个强大的工具来探测功能区域和残基的UAL，这将广泛影响酶的结构和功能的描述，在所有的生物素依赖性酶。 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。尿素酰胺解酶（UAL）通过启动允许生物体从单细胞酵母转变为多细胞丝状菌丝的形态学转换而促成致病性。由于UAL仅存在于细菌和真菌的一个子集中，因此它代表了对抗念珠菌感染的有希望和特异性靶标。本计画将利用X光晶体学、突变学及酵素动力学来探讨尿素酰胺解酵素之结构与功能。实现该项目的目标将导致UAL结构和功能的描述大大增强，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.