Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Incubation of [1-13C]-5-phosphoribosyl pyrophosphate ([1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of [1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik [Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway.     

Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton–Marshall assay (BMA) for diazotizable amines. The ZnSO₄ stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods.     

Crystal structure of Bacillus anthracis phosphoglucosamine mutase, an enzyme in the peptidoglycan biosynthetic pathway

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme that participates in the cytoplasmic steps of peptidoglycan biosynthesis. As peptidoglycan is essential for bacterial survival and is absent in humans, enzymes in this pathway have been the focus of intensive inhibitor design efforts. Many aspects of the structural biology of the peptidoglycan pathway have been elucidated, with the exception of the PNGM structure. We present here the crystal structure of PNGM from the human pathogen and bioterrorism agent Bacillus anthracis. The structure reveals key residues in the large active site cleft of the enzyme which likely have roles in catalysis and specificity. A large conformational change of the C-terminal domain of PNGM is observed when comparing two independent molecules in the crystal, shedding light on both the apo- and ligand-bound conformers of the enzyme. Crystal packing analyses and dynamic light scattering studies suggest that the enzyme is a dimer in solution. Multiple sequence alignments show that residues in the dimer interface are conserved, suggesting that many PNGM enzymes adopt this oligomeric state. This work lays the foundation for the development of inhibitors for PNGM enzymes from human pathogens.     

The interaction of monofluorofumarate with adenylosuccinate lyase

Monofluorofumarate was tested as an alternate substrate and inhibitor for adenylosuccinate lyase. Monofluorofumarate was found to be a slow reacting substrate when either AMP or AICAR (5-aminoimidazole 4-carboxamide ribonucleotide) were used as substrate acceptor molecules at pH 7.5. There was no indication that monofluorofumarate could induce the inactivation of adenylosuccinate lyase. The initial reaction product when monofluorofumarate was incubated with AMP in the presence of adenylosuccinate lyase has been determined to be 2-fluoro-adenylosuccinate. This molecule lost HF spontaneously, and the subsequent intermediate was rapidly hydrolyzed to oxalacetate and AMP. A similar reaction scheme was also observed when AICAR was utilized as a cosubstrate with monofluorofumarate. The initial reaction rate when 1.0 mM monofluorofumarate and 1.0 mM AMP were used as substrates with adenylosuccinate lyase was only 1.4% of the rate when 1.0 mM fumarate was used. AICAR (1.0 mM) was found to react with monofluorofumarate at 8.9% of the rate that it reacts with fumarate.     

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.     

Abnormal regulation of de novo purine synthesis and purine salvage in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase.

The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.     

The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds

Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway. Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism. Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features. The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution. Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features. Surprisingly, the structure of adenylosuccinate lyase from P. aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation. The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P. aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation. In addition, the protein is shorter in P. aerophilum than it is in other organisms. This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region.     

Crystallization and preliminary structural analysis of Bacillus subtilis adenylosuccinate lyase, an enzyme implicated in infantile autism.

Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X-ray diffraction is in progress. ASL is a 200-kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the beta-elimination of fumarate. A single point mutation in the human ASL gene has been linked to mental retardation with autistic features. In addition, ASL plays an important role in the bioprocessing of anti-HIV therapeutics. B subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K. The space group is P6(1)22 or P6(5)22 with a = b = 129.4 A; the length of the c-axis varies between 275 and 290 A, depending on the crystal. An analysis of solvent content indicates a dimer in the asymmetric unit, although a self-rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes. Structure determination by isomorphous replacement is in progress.     

Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.     

Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of analogues and key derivatives of 10-CF3CO-DDACTHF as inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Polyglutamate analogues of 1 were evaluated as inhibitors of Escherichia coli and recombinant human (rh) GAR Tfase, and AICAR Tfase. Although the pentaglutamate 6 was found to be the most active inhibitor of the series tested against rhGAR Tfase (Ki=0.004 microM), little distinction between the mono-pentaglutamate derivatives was observed (Ki=0.02-0.004 microM), suggesting that the principal role of the required polyglutamation of 1 is intracellular retention. In contrast, 1 and its defined polyglutamates 3-6 were much less inactive when tested against rhAICAR Tfase (Ki=65-0.120 microM) and very selective (> or =100-fold) for rh versus E. coli GAR Tfase. Additional key analogues of 1 were examined (7 and 8) and found to be much less active (1000-fold) highlighting the exceptional characteristics of 1.     

The crystal structure of the first enzyme in the pantothenate biosynthetic pathway,ketopantoate hydroxymethyltransferase,from M tuberculosis

Ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first committed step in the biosynthesis of pantothenate, which is a precursor to coenzyme A and is required for penicillin biosynthesis. The crystal structure of KPHMT from Mycobacterium tuberculosis was determined by the single anomalous substitution (SAS) method at 2.8 A resolution. KPHMT adopts a structure that is a variation on the (beta/alpha) barrel fold, with a metal binding site proximal to the presumed catalytic site. The protein forms a decameric complex, with subunits in opposing pentameric rings held together by a swapping of their C-terminal alpha helices. The structure reveals KPHMT's membership in a small, recently discovered group of (beta/alpha) barrel enzymes that employ domain swapping to form a variety of oligomeric assemblies. The apparent conservation of certain detailed structural characteristics suggests that KPHMT is distantly related by divergent evolution to enzymes in unrelated pathways, including isocitrate lyase and phosphoenolpyruvate mutase.     

Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis

Summary The gene-enzyme relationship has been established for most of the steps of the purine de novo biosynthetic pathway in Bacillus subtilis. The synthesis of inosine monophosphate (IMP) involves ten steps, and the branching from IMP to AMP and to guanosine monophoshate (GMP) synthesis both require two steps. To avoid confusion in the nomenclature of the pur genes we have adopted the Escherichia coli system for B. subtilis. The two genes specifying the enzymes catalysing the conversion of IMP to succinyl-AMP (purA), and the conversion of IMP to xanthosine monophosphate (guaB), occur as single units whilst the other purine genes are clustered at 55 degrees on the B. subtilis linkage map. Based on transformation and transduction studies, and on complementation studies using B. subtilis pur genes cloned in plasmids, the arrangement of some of the clustered genes has been determined relative to outside markers. The following gene order has been established: pbuG-purB-purF-purM-purH-purD-tre. Three other genes were also found to be located in the cluster, guaA, purL and purE/C. However, we were not able to find their exact location. When the purF, purM, purD and purB genes of B. subtilis are present in plasmids they are capable of directing the synthesis in E. coli of phosphoribosylpyrophosphate amidotransferase (purF), aminoimidazole ribonucleotide synthetase (purM), glycinamide ribonucleotide synthetase (purD) and adenylosuccinate lyase (purB), respectively.     

The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but l-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.     

A gene correcting the defect in the CHO mutant Ade -H, deficient in a branch point enzyme (adenylosuccinate synthetase) of de novo purine biosynthesis, is located on the long arm of chromosome 1

Somatic hybrids between human cells and the Chinese hamster ovary (CHO) K1 mutant, Ade -H cells, were selected for purine prototrophy by growth in adenine-free medium. The Ade -H mutant is defective in the enzyme adenylosuccinate (AMPS) synthetase (ADSS; EC 6.3.4.4), which carries out the first of a two-step sequence in the biosynthesis of AMP from IMP, and therefore requires exogenous adenine for growth. The presence of the long arm of human chromosome 1 in the hybrids is 100% concordant for the ability to grow in adenine-free medium and restoration of the enzyme activity. Hybrid segregants that lose the ability to grow in adenine-free medium lose all or a portion of chromosome 1 and enzyme activity. Southern blot hybridization with a chromosome 1-specific probe, BCMI, confirms the existence of human chromosome 1 in these hybrids. Analysis of a human/CHO translocation chromosome that arose in one of the hybrids suggests that the gene correcting the defect lies in the region 1 cen-1q12. In summary, we have shown by cytogenetics, segregant analysis, biochemical assay, and Southern blot analysis that human chromosome 1, most likely in the region 1cen-1q12, corrects the defect in ADSS-deficient mutant Ade-H cells.     

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.     

Purification and properties of an enzyme duet, phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase, involved in the biosynthesis of purine nucleotides de novo (Short Communication)

Purification and some properties of an adjacent pair of enzymes in the biosynthetic pathway to AMP, phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6), are described. The enzymes form a duet, which may be regarded as a single molecule with a dual function, or as two very similar substances.