BIOCHEMICAL ROLE OF THE CRYPTOCOCCUS-NEOFORMANS ADE2 PROTEIN IN FUNGAL DE-NOVO PURINE BIOSYNTHESIS

Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxyla ses from Escherichia coli and Gallus gallus have identified this centr al step in de novo purine biosynthesis as a case for unusual divergenc e in primary metabolism, Recent discoveries establish the fungal AIR c arboxylase, encoded by the ADE2 gene, as essential for virulence in ce rtain pathogenic organisms, This investigation is a biochemical analys is that links the fungal ADE2 protein to the function of the E. coli A IR carboxylase system, A cDNA clone of ADE2 from Cryptococcus neoforma ns was isolated by genetic complementation of a purE-deficient. strain of E. coli. High-level expression of the C. neoformans ADE2 was achie ved, which enabled the production and purification of AIR carboxylase, Amino acid sequence alignments, C-terminal deletion mutants, and bioc hemical assays indicate that the ADE2 enzyme is a two-domain, bifuncti onal protein, The N-terminal domain is related to E. coil PurK and a s eries of kinetic experiments show that the ADE2-PurK activity uses AIR , ATP, and HCO3- as substrates, The biosynthetic product of the ADE2-P urK reaction was identified as N-5-carboxyaminoimidazole ribonucleotid e (N-5-CAIR) by H-1 NMR, thus confirming that the C-terminal domain co ntains a catalytic activity similar to that of the E. coil PurE. By us ing an in situ system for substrate production, the steady-state kinet ic constants for turnover of N-5-CAIR by ADE2 were determined and toge ther with stoichiometry measurements, these data indicate that ADE2 ha s a balance in the respective catalytic turnovers to ensure efficient flux. Distinctive features of the PurE active site were probed using 4 -nitro-5-aminoimidazoIe ribonucleotide (NAIR), an analog of the produc t 4-carboxy-5-aminoimidazole ribonucleotide (CAIR), NAIR was shown to be a selective inhibitor of the ADE2-PurE activity (K-i = 2.4 mu M), w hereas it is a slow-binding inhibitor of the G. gallus enzyme which fu rther distinguishes the fungal ADE2 from the G. gallus AIR carboxylase . As such, this enzyme represents a novel intracellular target for the discovery of antifungal agents. (C) 1998 Academic Press.