Structural and Biochemical Investigation of UTP Cyclohydrolase

The URC (uracil catabolism) 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, followed by pyrimidine ring cleavage catalyzed by Urc1p, a distant homologue of GTP cyclohydrolase II (RibA). Here we report a biochemical investigation of Urc1p from Pichia pastoris (Komagataella phaffii) (PpUrc1p) and its bacterial homologue from Rhodococcus wratislaviensis (RwUrcA). Like RibA, the substrate of recombinant PpUrc1p was found to be a nucleoside triphosphate (UTP), and turnover was accompanied by the release of pyrophosphate. The products phosphoribosylurea and malonic semialdehyde were confirmed by mass spectrometry. We also determined a 1.60 Å crystal structure of RwUrcA, which included a C-terminal segment that is not resolved in RibA structures reported to date, containing a conserved Lys residue positioned to interact with the UTP α-phosphate. Further mutagenesis studies revealed roles of active site residues in substrate binding and catalysis, providing insights into the mechanism by which Urc1p catalyzes two sequential hydrolyses at C6 and C4 for the uracil ring.