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S M Firestine

Publications and source records attributed to S M Firestine.

5 recordsLinked to original sources
PubMed1998

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.

Adenosine Triphosphate↗
PubMed1996

Threading your way to protein function.

It is still very difficult to determine the function of a protein from its sequence. One potential solution to the problem combines the concept of enzyme superfamilies with modern methods of protein structure prediction. Active-site templates can be used as search tools to identify new members of the superfamilies.

Amino Acid Sequence↗
PubMed1994

Carboxylases in de novo purine biosynthesis. Characterization of the Gallus gallus bifunctional enzyme.

Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes. In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR. This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992). A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure. Selective assays have been developed for all the enzymatic activities of the bifunctional protein. The G. gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E. coli, supporting the hypothesis that the two enzymes require different substrates. No common cofactors or metals are required for catalysis. Each catalytic activity has been shown to be independent by selective inactivation of SAICAR synthetase with the affinity agent 5'-[4-(fluorosulfonyl)benzoyl]-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR). The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site.

Adenosine↗
PubMed1994

Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms.

A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E. coli and G. gallus has been conducted using in vivo and in vitro studies. In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. The E. coli PurE and the C-terminal domain of the G. gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities. The substrate requirements of AIR carboxylases from E. coli and G. gallus have been compared by a series of in vitro experiments. The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E. coli PurE (Mueller et al., 1994) but not for the G. gallus AIR carboxylase. In contrast, AIR and CO2 are substrates for the G. gallus AIR carboxylase. The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR). NAIR is a tight-binding inhibitor of the G. gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E. coli PurE. These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoimidazole Carboxamide↗