Biochemical Role of theCryptococcus neoformansADE2 Protein in Fungalde novoPurine Biosynthesis

Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxylases fromEscherichia coliandGallus gallushave identified this central step inde novopurine biosynthesis as a case for unusual divergence in primary metabolism. Recent discoveries establish the fungal AIR carboxylase, encoded by theADE2gene, as essential for virulence in certain pathogenic organisms. This investigation is a biochemical analysis that links the fungal ADE2 protein to the function of theE. coliAIR carboxylase system. A cDNA clone ofADE2fromCryptococcus neoformanswas isolated by genetic complementation of apurE-deficient strain ofE. coli.High-level expression of theC. neoformans ADE2was achieved, which enabled the production and purification of AIR carboxylase. Amino acid sequence alignments, C-terminal deletion mutants, and biochemical assays indicate that the ADE2 enzyme is a two-domain, bifunctional protein. The N-terminal domain is related toE. coliPurK and a series of kinetic experiments show that the ADE2–PurK activity uses AIR, ATP, and HCO3−as substrates. The biosynthetic product of the ADE2–PurK reaction was identified asN5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by1H NMR, thus confirming that the C-terminal domain contains a catalytic activity similar to that of theE. coliPurE. By using anin situsystem for substrate production, the steady-state kinetic constants for turnover ofN5-CAIR by ADE2 were determined and together with stoichiometry measurements, these data indicate that ADE2 has a balance in the respective catalytic turnovers to ensure efficient flux. Distinctive features of the PurE active site were probed using 4-nitro-5-aminoimidazole ribonucleotide (NAIR), an analog of the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). NAIR was shown to be a selective inhibitor of the ADE2–PurE activity (Ki= 2.4 μM), whereas it is a slow-binding inhibitor of theG. gallusenzyme which further distinguishes the fungal ADE2 from theG. gallusAIR carboxylase. As such, this enzyme represents a novel intracellular target for the discovery of antifungal agents.