Structural And Biochemical Investigation Of Key Enzymes In The Uracil Catabolism Pathway

Pyrimidines are nitrogen-containing heterocyclic aromatic compounds and components of nucleic acids,and are thus ubiquitous in nature.Microbes use pyrimidines as carbon or nitrogen source to support growth.To date,four pyrimidine degradative pathways have been described,namely the reductive（PYD）,oxidative,Rut and URC pathways,each employing a different strategy for the cleavage of the stable aromatic pyrimidine ring.While all four pathways are found in bacteria,only the PYD and URC pathways are found in eukaryotes.The URC（uracil catabolism）pathway is the most recently described pyrimidine degradation pathway and,from an enzymology perspective,the least well understood.The URC pathway,present in fungi and bacteria,enables pyrimidines to be used as nitrogen sources for growth.Although its mechanistic details are unclear,uracil is thought to be first converted into a uridine nucleotide by Urc6p,followed by pyrimidine ring cleavage catalyzed by Urc1p（a distant homolog of GTP cyclohydrolase II（Rib A））,producing 5-phosphoribosylurea and malonic semialdehyde.5-phosphoribosylurea was converted by Urc4p（domain of unknown function,DUF1688 family）,generating phosphate ribose and urea,followed by Urc3p,5p-catalyzed urea degradation to release 2 NH₃ and CO₂.Malonic semialdehyde was reduced by Urc8p forming 3-hydroxypropionic acid（3HPA）.In this thesis,we focus on Urc1p and Urc4p,which are unique to the URC pathway and have not been biochemically and structurally characterized.The remaining enzymes of the URC pathway are better understood,as they have been reported in other nucleotide salvage or nitrogen metabolism pathways.Like Rib A,the substrate of recombinant Pp Urc1p was found to be a nucleoside triphosphate（UTP）,and turnover was accompanied by release of pyrophosphate.Formation of the products,phosphoribosylurea and malonic semialdehyde,was confirmed by mass spectrometry.RwUrcA was then crystallized and its structure was solved by X-ray diffraction at 1.60?resolution.This is the first complete structure of a protein in the GTP cyclohydrolase II family.Previously,the crystal structure of E.coli Rib A has been reported.However,the C-terminal segment（175-196 aa）was missing in that structure.Based on our structure,this part of the protein also contributes to forming the active site.A conserved Lys residue was found in our structure positioned to interact with the UTPα-phosphate.Formation of the covalent Urc1p-UMP intermediate,coupled to the energetically favorable pyrophosphate release,may drive substrate binding in a conformation that predisposes it to reaction,thus accelerating the cleavage of the otherwise hydrolytically inert uracil ring.Further mutagenesis studies revealed roles of active site residues including this lysine residue in substrate binding and catalysis,providing insights into the mechanism,by which Urc1p catalyzes two sequential hydrolyses at C6 and C4 of the uracil ring.In addition,this thesis also reports the recombinant production and enzymatic assays of Pp Urc4p,which was found capable of converting phosphoribosylurea to urea and phosphate ribose,detected by mass spectrometry.In the future,we will do further biochemical and structural investigation on Urc4p.Taken together,we for the first time demonstrated the activities of two key enzymes in the previously proposed URC pathway and investigated the catalytic mechanism of Urc1p in great details.