Biochemical role of the Cryptococcus neoformans ADE2 protein in fungal de novo purine biosynthesis.
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.
Firestine
S M
SM
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333, USA.
Misialek
S
S
Toffaletti
D L
DL
Klem
T J
TJ
Perfect
J R
JR
Davisson
V J
VJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003455:Cryptococcus neoformans / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D011687:Purines / Q000096:biosynthesis*; D012265:Ribonucleotides / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001639:Bicarbonates; D029968:Escherichia coli Proteins; D011687:Purines; D012265:Ribonucleotides; C024828:aminoimidazole ribotide; D000620:Aminoimidazole Carboxamide; D000255:Adenosine Triphosphate; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; C075015:purK protein, E coli; C031143:AICA ribonucleotide