Aminoimidazole Ribonucleotide Research Articles

The Direct Precursor of the Pyrimidine Moiety of Thiamin Is Not Urocanic Acid but Histidine in Saccharomyces cerevisiae

The biosynthetic route of the pyrimidine moiety of thiamin is different in prokaryotes and eukaryotes. In prokaryotes, the pyrimidine moiety is synthesized from aminoimidazole ribonucleotide, an intermediate of purine biosynthesis, while in eukaryotes, we have reported that the N-1, C-2, and N-3 atoms of the imidazole ring of histidine are incorporated into N-3, C-4, and the amino group attached to the C-4 atoms of the pyrimidine moiety, respectively, as a unit; the rest of the atoms of the pyrimidine moiety originate from pyridoxine as a unit. It has been reported that urocanic acid, the deaminated compound of histidine, is the direct precursor of the pyrimidine moiety. In the present report, we have investigated whether histidine or urocanic acid is the direct precursor of the pyrimidine moiety in Saccharomyces cerevisiae, using tracer experiments with 1) (13)C-formate and urocanic acid, 2) (15)N-NH(4)Cl and urocanic acid, 3) (15)N-NH(4)Cl and histidine, and 4) (13)C-histidine and urocanic acid. The GC-MS analysis revealed that the incorporation of the (15)N atom of (15)NH(4)Cl was not affected by the presence of urocanic acid, although it was affected by histidine, and the incorporation of (13)C-histidine was not affected by the presence of urocanic acid. These results confirm that histidine is the direct precursor of the pyrimidine moiety of thiamin in S. cerevisiae.

In situ proteolysis for protein crystallization and structure determination.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

N5-CAIR Mutase: Role of a CO2 Binding Site and Substrate Movement in Catalysis,

N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.

Octameric Structure of the Human Bifunctional Enzyme PAICS in Purine Biosynthesis

Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an important bifunctional enzyme in de novo purine biosynthesis in vertebrate with both 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-( N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase (SAICARs) activities. It becomes an attractive target for rational anticancer drug design, since rapidly dividing cancer cells rely heavily on the purine de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Here, we report the crystal structure of human PAICS, the first in the entire PAICS family, at 2.8 Å resolution. It revealed that eight PAICS subunits, each composed of distinct AIRc and SAICARs domains, assemble a compact homo-octamer with an octameric-carboxylase core and four symmetric periphery dimers formed by synthetase domains. Based on structural comparison and functional complementation analyses, the active sites of SAICARs and AIRc were identified, including a putative substrate CO 2-binding site. Furthermore, four symmetry-related, separate tunnel systems in the PAICS octamer were found that connect the active sites of AIRc and SAICARs. This study illustrated the octameric nature of the bifunctional enzyme. Each carboxylase active site is formed by structural elements from three AIRc domains, demonstrating that the octamer structure is essential for the carboxylation activity. Furthermore, the existence of the tunnel system implies a mechanism of intermediate channeling and suggests that the quaternary structure arrangement is crucial for effectively executing the sequential reactions. In addition, this study provides essential structural information for designing PAICS-specific inhibitors for use in cancer chemotherapy.

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase ( AK) and adenine phosphoribosyltransferase ( APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase ( GART) and 5-aminoimidazole ribonucleotide synthetase ( AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5′-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho- d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8- 14C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.

Nucleotide Complexes of Escherichia coli Phosphoribosylaminoimidazole Succinocarboxamide Synthetase

Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP.CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg(2+), two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the alpha- and beta-phosphoryl groups of ADP. The ADP.CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204-221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms.

Risk genotypes in folate‐dependent enzymes and their association with methotrexate‐related side effects in rheumatoid arthritis

Methotrexate (MTX) is an antifolate agent that is often associated with toxicity. This study investigated whether risk genotypes for folate-dependent enzymes are associated with the toxicity of MTX in patients with rheumatoid arthritis (RA). Blood was collected for analysis in a muticenter, cross-sectional study of RA patients who had been receiving MTX for at least 1 month prior to enrollment, and side effects occurring at the time of a single study visit were recorded. Low-penetrance risk genotypes in methylenetetrahydrofolate reductase (MTHFR) 677TT, thymidylate synthase (TSER) *2/*2 (variable number of tandem repeats), amino imidazole ribonucleotide transformylase (ATIC) 347GG, and serine hydroxymethyltransferase (SHMT1) 1420CC were measured and summed to constitute the toxicogenetic index specific to each patient. Statistical analyses consisted of logistic regression models with clustered-center effects, and associations with risk genotypes were expressed as adjusted odds ratios (ORs). Among 214 patients enrolled at 4 study sites, a total of 67 patients (31%) presented with a side effect (gastrointestinal event, headache, lethargy, alopecia, cough, or dyspnea). Risk genotypes associated with side effects in the central nervous system were MTHFR 677TT (OR 3.3, P < 0.01) and SHMT1 1420CC (OR 2.4, P < 0.05). ATIC 347GG was associated with gastrointestinal side effects (OR 3.0, P < 0.01), while TSER*2/*2 (OR 5.4, P < 0.01) and SHMT1 1420CC (OR 3.2, P < 0.01) were associated with alopecia. The toxicogenetic index ranged from 0 to 3 (median 1). An index of 3 was associated with an approximately 7-fold higher likelihood of having a side effect compared with an index of 0 (P < 0.01). These data suggest that a composite index of the cumulative risk genotypes in folate-dependent enzymes may be an effective means of profiling RA patients who develop side effects to MTX.

Crystal structure of PurE (BA0288) from Bacillus anthracis at 1.8 Å resolution

Crystal structure of PurE (BA0288) from <i>Bacillus anthracis</i> at 1.8 Å resolution

Biosynthesis of the thiamin pyrimidine: the reconstitution of a remarkable rearrangement reaction.

The conversion of 5-aminoimidazole ribonucleotide (AIR) into 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) is a fascinating reaction on the thiamin biosynthetic pathway in bacteria and is probably the most complex unresolved rearrangement in primary metabolism. We have successfully reconstituted this reaction in a cell-free system. The E. coli thiC gene product and an additional unidentified E. coli protein are required for the reaction. In addition, SAM and nicotinamide cofactors are required for full activity. Labeling studies to determine the origin of most of the atoms in the pyrimidine are described. Based on these studies, a new mechanism for HMP formation is proposed.

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea.

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.

Effect of short-term N(2) deficiency on expression of the ureide pathway in cowpea root nodules.

Root systems of 28-d-old cowpea (Vigna unguiculata L. Walp cv Vita 3: Bradyrhizobium sp. strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O(2) (80:20, v/v) for 48 h and then returned to air. Root systems of control plants were maintained in air throughout. Nodules were harvested at the same times in control and Ar:O(2)-treated root systems. Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies. AIRS activity and protein both declined to very low levels within 6 h in Ar:O(2) together with a decline in transcript level of pur5, the encoding gene. GART activity, protein, and transcript (pur3) levels were relatively stable. Uricase activity declined in Ar:O(2) as rapidly as AIRS activity but the protein was stable. PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable. The data indicate that the flux of fixed N from bacteroids (N(2)-fixing nodule bacteria) is in some way associated with transcriptional control over pur5 and possibly also catabolism of AIRS protein. In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity. The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.

A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application

We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also contructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrataADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrataCEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi.

Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase.

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.

The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway.

Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.

Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway.

Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE. PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR. PurEs from higher eukaryotes are homologous to E. coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly. The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry. A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit. The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit. Co-crystallization experiments with CAIR confirm this to be the mononucleotide-binding site. The nucleotide is bound predominantly to one subunit, with conserved residues from a second subunit making up one wall of the cleft. The crystal structure of PurE reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of PurE with CAIR, reveals the location of the active site. The environment of the active site and the analysis of conserved residues between the two classes of PurEs suggests a model for the differences in their substrate specificities and the relationship between their mechanisms.

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily.

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.

Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [(14)C]-p-fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1). The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase. After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured. All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent. The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group. The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., Weaver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests additional flexibility in the ATP binding region.

Evidence for the direct transfer of the carboxylate of N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) to generate 4-carboxy-5-aminoimidazole ribonucleotide catalyzed by Escherichia coli PurE, an N5-CAIR mutase.

Formation of 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) in the purine pathway in most prokaryotes requires ATP, HCO3-, aminoimidazole ribonucleotide (AIR), and the gene products PurK and PurE. PurK catalyzes the conversion of AIR to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) in a reaction that requires both ATP and HCO3-. PurE catalyzes the unusual rearrangement of N5-CAIR to CAIR. To investigate the mechanism of this rearrangement, [4,7-13C]-N5-CAIR and [7-14C]-N5-CAIR were synthesized and separately incubated with PurE in the presence of ATP, aspartate, and 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetase (PurC). The SAICAR produced was isolated and analyzed by NMR spectroscopy or scintillation counting, respectively. The PurC trapping of CAIR as SAICAR was required because of the reversibility of the PurE reaction. Results from both experiments reveal that the carboxylate group of the carbamate of N5-CAIR is transferred directly to generate CAIR without equilibration with CO2/HCO3- in solution. The mechanistic implications of these results relative to the PurE-only (CO2- and AIR-requiring) AIR carboxylases are discussed.

A high-copy-number ADE2-bearing plasmid for transformation of Candida glabrata.

The Candida glabrata ADE2 gene encoding aminoimidazole ribonucleotide (AIR) carboxylase (EC 4.1.1.21) was isolated by complementation of the ade2-1 mutation in Saccharomyces cerevisiae. The predicted amino acid (aa) sequence is 75% identical to that of S. cerevisiae. Integrative transformation was used to produce a C. glabrata strain bearing a deletion of ADE2 coding sequences. A high-copy-number shuttle vector bearing the ADE2 gene was constructed and contains a fragment of S. cerevisiae mitochondrial (mt) DNA that confers the ability to replicate autonomously in C. glabrata.

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.