Urea amidolyase (UAL) is a key virulence factor that regulates the yeast to hyphae switch in the opportunistic pathogen, Candida albicans. UAL is a multi-domain enzyme with two enzyme activities: urea carboxylase (UC) and allophanate hydrolase (AH). UC is a biotin-dependent carboxylase that adds a carboxyl group to urea to make allophanate by the coordinated action of three domains. Allophanate is subsequently hydrolyzed into NH 3 and CO2 in the AH domain. Studies on the structure and function of UAL may lead to treatments for systemic candidiasis and can serve to clarify the molecular basis for multi-functional swinging arm enzymes.
In the present study, the first structure of AH was solved by X-ray crystallography. Site-directed mutagenesis and steady-state kinetic analysis of Granulibacter bethesdensis AH reveal a role for two residues, Tyr299 and Arg307, in maintaining substrate stringency and providing transition state stabilization. In addition, as UAL activity is essential for urea-dependent growth of yeast, a yeast genetic screen was developed to identify key functional residues in UAL. Random mutations were introduced in the targeted region of UAL, and the roles of an active site loop and distant residues in catalysis were highlighted in AH. The mechanisms of intermediate transfer between UC and AH were also studied. To investigate whether allophanate is channeled between the active sites, co-purification studies and substrate channeling assays were performed in vitro to detect stable or transient interactions between UC and AH. No strong coupling was detected between UC and AH. In addition, the coupling efficiency between the individual catalytic domains in UC is low, indicating that an as yet undiscovered activator may serve to facilitate the coordination among the three catalytic domains of UC. Taken together, these studies describe UAL as a complex, multi-functional enzyme that exhibits a high degree of substrate specificity in each domain, but does not require efficient substrate channeling to accomplish catalysis. These descriptions serve to advance the understanding of the mechanism of multi-functional enzymes as a whole.
|Advisor:||St. Maurice, Martin|
|Commitee:||Fu, Jianhua, Noel, Dale, Stuart, Rosemary, Yang, Pinfen|
|School Location:||United States -- Wisconsin|
|Source:||DAI-B 76/04(E), Dissertation Abstracts International|
|Keywords:||Enzymology, Genetics, Structural biology|
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