Investigation of the structure and function of urea amidolyase

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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.