Abstract:
:Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxylases from Escherichia coli and Gallus gallus have identified this central step in de novo purine biosynthesis as a case for unusual divergence in primary metabolism. Recent discoveries establish the fungal AIR carboxylase, encoded by the ADE2 gene, as essential for virulence in certain pathogenic organisms. This investigation is a biochemical analysis that links the fungal ADE2 protein to the function of the E. coli AIR carboxylase system. A cDNA clone of ADE2 from Cryptococcus neoformans was isolated by genetic complementation of a purE-deficient strain of E. coli. High-level expression of the C. neoformans ADE2 was 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 to E. coli PurK 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 as N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by 1H NMR, thus confirming that the C-terminal domain contains a catalytic activity similar to that of the E. coli PurE. By using an in situ system for substrate production, the steady-state kinetic constants for turnover of N5-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 (K1 = 2.4 microM), whereas it is a slow-binding inhibitor of the G. gallus enzyme which further 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.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Firestine SM,Misialek S,Toffaletti DL,Klem TJ,Perfect JR,Davisson VJdoi
10.1006/abbi.1997.0512subject
Has Abstractpub_date
1998-03-01 00:00:00pages
123-34issue
1eissn
0003-9861issn
1096-0384pii
S0003-9861(97)90512-9journal_volume
351pub_type
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