A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.     

PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.     

Assignment of a third purine biosynthetic gene (glycinamide ribonucleotide transformylase) to human chromosome 21.

Using a series of human-hamster hybrid cell lines, a gene coding for glycinamide ribonucleotide transformylase was mapped to human chromosome 21. The availability of hybrids containing only portions of chromosome 21 allowed the gene to be assigned to the region between the q11.2 and the q22.2 bands, inclusive. Differentiation of human and hamster glycinamide ribonucleotide transformylase was accomplished via an immunoprecipitation assay that employed a polyclonal antibody raised against the human enzyme.     

De novo purine ribonucleotide biosynthesis in adult Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

1. Radioactivity was found to be associated with purine ribonucleotides in extracts of gravid adult Angiostrongylus cantonensis from the lungs of rats when the parasite was incubated in a medium containing [14C]glycine. 2. The rate of incorporation was at least 0.2 pmole/min per mg of parasite and approximately equal amounts of adenine and guanine ribonucleotides were synthesized. 3. This is the first demonstration of the capacity for a mammalian parasitic helminth to synthesize purines de novo.     

De novo purine nucleotide biosynthesis: cloning, sequencing and expression of a chicken PurH cDNA encoding 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase

The purH cDNA, encoding 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) transformylase-inosine monophosphate cyclohydrolase (ATIC), was cloned by functional complementation of an Escherichia coli purH mutant using a chicken liver cDNA expression library. This represents the first report of the cloning of any eukaryotic ATIC-encoding cDNA (PurH). The avian ATIC mRNA is 2.3 kb long and encodes a protein with an Mr of 64,422. The deduced amino acid sequence is 36% identical to the bacterial purH-encoded enzymes from Bacillus subtilis and E. coli. The avian cDNA was expressed as a glutathione S-transferase (GST) fusion protein that was purified in a single step by affinity chromatography. A novel vector was employed which permits rapid and highly efficient cleavage of the GST fusion protein yielding 10 mg of purified PurH product per liter of bacterial culture. Km values were determined with the purified fusion protein utilizing AICAR and (6-R)N10-formyl-tetrahydrofolate as substrates. These values compare favorably with the isolated avian enzyme, supporting the idea that kinetic, as well as other physical properties of the recombinant fusion protein are similar to the native avian enzyme. Large quantities of purified enzyme and the ability to generate site-directed mutations should make mechanistic studies possible. The recombinant enzyme also affords a simple and reliable approach to identifying new antifolates.     

Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies.

Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.     

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO₂ and is dispensable in the presence of high CO₂ concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO₂-binding activity.     

De novo purine biosynthesis in the crustacean Artemia: Influence of salinity and geographical origin

Summary In vivo studies of the incoporation of [U-¹⁴C]glycine into purine nucleotides have established the de novo pathway for purine biosynthesis in Artemia sp. during the early period of larval development. This pathway can be modified by the salt concentration of the incubation media. In addition, Artemia of different geographical origins may differ with respect to the detection, functionality and variability of this metabolical pathway.Abbreviations ADP adenosine, diphosphate - ASN acid soluble nucleotides - ATP adenosine triphosphate - DNA desoxyribonucleic acid - GDP guanosine diphosphate - GP₄G pl, p4-diguanosine 5-tetraphosphate - HPLC high performance liquid chromatography - PCA perchloric acid - RNA ribonucleic acid     

A quantum chemical study of the water-assisted mechanism in one-carbon unit transfer reaction catalyzed by glycinamide ribonucleotide transformylase

A theoretical study for the water-assisted mechanism in one-carbon unit transfer reaction catalyzed by glycinamide ribonucleotide transformylase (GAR Tfase) is investigated in which the proton transfers in an indirect way and the energy barrier for each transition state has been lowered about 80–100 kJ/mol when compared with the corresponding one in a no-water-involved mechanism. There are two possible pathways in each mechanism: one is concerted and the other is stepwise. Our results have verified the presumption from experiments that one water molecule can assist to achieve the whole reaction. Because the addition of this water molecule in the transition states can relax the strong strain in the unstable system and greatly lowered the energy barrier. The water-assisted paths are preferable to the no-water-involved ones and the bulk solvent effect of water is also discussed.     

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.     

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.     

Inhibition of de novo purine biosynthesis and interconversion by 6-methylpurine in Escherichia coli

The inhibition of Escherichia coli strain B and strain W-11 by 6-methylpurine depended on the formation of 6-methylpurine ribonucleotide by the action of adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). 6-Methylpurine ribonucleotide inhibited the de novo synthesis of purines, presumably via pseudofeedback inhibition of phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14). The same mechanism accounted for its inhibition of adenylosuccinate synthetase [IMP: l-aspartate ligase (GDP), EC 6.3.4.4]. Adenine and 6-methylaminopurine prevented inhibition by competing for the action of adenine phosphoribosyltransferase. In addition, adenine reversed this inhibition by replenishing the AMP to bypass both sites of inhibition. Nonproliferating suspensions of strain B-94, which lacked adenylosuccinate lyase (EC 4.3.2.2), converted exogenous hypoxanthine and aspartate to succinoadenine derivatives which accumulated in the medium. Compounds which inhibited adenylosuccinate synthetase inhibited accumulation of the succinoadenine derivatives. A method was described for the isolation of mutants which potentially possessed an altered adenylosuccinate synthetase.     

De novo biosynthesis of FSH like peptide by the human prostate

Human benign prostatic hyperplasia tissue is able to synthesize immunoreactive FSH (IR FSH) in vitro. The prostatic FSH is similar to pituitary FSH as evident by co-elution on Sephadex G-100. High performance gel filtration chromatography and Western blot analysis. Immunocytochemical localization studies indicate positive staining in the cytoplasm of prostatic epithelial cells. There is a two fold increase in the concentration of immunoreactive FSH in benign hyperplastic tissue as compared to normal prostate.     

ACTH stimulation of purine nucleotide biosynthesis in the adrenal cortex. ACTH stimulation of purine biosynthesis

In the presence of azaserine an inhibitor of phosphoribosylformylglycineamidine synthetase (EC 6.3.5.3) the incorporation of [2-¹⁴C]glycine into 5′-phosphoribosylglycineamide and its formyl derivative was measured in 105,000g supernatant fraction prepared from a homogenate of adrenal cortex. Corticotropin at a level of 1-0.001 nm markedly stimulated in 10 min these early steps of purine biosynthesis. The stimulus was in addition to that achieved with added glucose-6-phosphate and NADP. Increased synthesis of precursors of purine nucleotides is due to ACTH activation of adrenal glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and thus the pentose cycle with an increase in 5′-phosphoribosylpyrophosphate. The generation of this latter compound is presumed to be a rate-limiting factor to 5′-phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) the first enzyme of de novo purine biosynthesis.     

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.     

Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12

5'-Phosphoribosylglycinamide transformylase (EC 2.1.2.2), encoded by the purN gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosylformylglycinamide from 5'-phosphoribosylglycinamide (GAR). The mature protein, as deduced from the purN structural gene sequence, contains 212 amino acid residues and has a calculated Mr of 23,241. The purN gene is located adjacent to and immediately downstream from the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase where the initiation codon for GAR transformylase overlaps the termination codon of AIR synthetase. Based on polarity studies, the expression of the purN gene originates from the purM control region and thus forms a purMN operon. The E. coli GAR transformylase shows greater homology to the GAR transformylase domain of the trifunctional Gart polypeptide of Drosophila than to the single GAR transformylase of Saccharomyces. Immediately downstream from the purN gene of the purMN operon is a region of dyad symmetry capable of forming a hairpin stem and loop structure characteristic of a rho-independent terminator.     

Molecular cloning of cDNAs encoding the human bombesin-like peptide neuromedin B. Chromosomal localization and comparison to cDNAs encoding its amphibian homolog ranatensin

The amidated decapeptide neuromedin B (NMB) is the mammalian homolog of the amphibian bombesin-like peptide ranatensin. cDNAs encoding human neuromedin B and amphibian ranatensin were isolated from human hypothalamic and Rana pipiens skin libraries, respectively. Sequence analysis revealed that NMB is encoded in a 76-amino acid precursor and ranatensin in an 82-amino acid precursor. In the NMB preprohormone, the sequence of the large form of NMB (NMB-22) immediately follows the signal peptide and is, in turn, followed by a dibasic cleavage site and a 17-amino acid carboxyl-terminal extension peptide. The structure for the ranatensin preprohormone is very similar. RNA blot analysis shows two NMB mRNA species, each approximately 800 bases, with wide distribution in brain and gastrointestinal tract. Genomic DNA blot analysis is consistent with a single human NMB gene. Analysis of mouse-human somatic cell hybrids indicates that this gene is localized on the long arm of human chromosome 15. Since the gene for human gastrin-releasing peptide is on chromosome 18, this analysis demonstrates that the bombesin-like peptide genes are not clustered.     

Heterospecific cloning of Arabidopsis thaliana cDNAs by direct complementation of pyrimidine auxotrophic mutants of Saccharomyces cerevisiae. I. Cloning and sequence analysis of two cDNAs catalysing the second,fifth and sixth steps of the de novo pyrimidine biosynthesis pathway

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to ch1B, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA ⁺ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA ⁺ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-ProHis-Cys-motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.     

Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

Successive rounds of chromosome-mediated gene transfer were used to complement a hamster cytidine auxotroph deficient in CTP synthetase activity and eventually to clone human genomic and cDNA fragments coding for the structural gene. Our approach was to isolate human Alu+ fragments from a tertiary transfectant and to utilize these fragments to screen a panel of primary transfectants. In this manner two DNA fragments, both mapping within the structural gene, were identified and used to clone a partial length cDNA. The remaining portion of the open reading frame was obtained through the RACE polymerase chain reaction technique. The open reading frame encodes 591 amino acids having a striking degree of similarity to the Escherichia coli structural gene (48% identical amino acids with 76% overall similarity including conservative substitutions) with the glutamine amide transfer domain being particularly conserved. As regulatory mutations of CTP synthetase confer both multi-drug resistance to agents widely used in cancer chemotherapy and a mutator phenotype, the cloning of the structural gene will be important in assessing the relevance of such phenotypes to the development of cellular drug resistance.