scholarly journals The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathione-β-synthase domain

2021 ◽  
Author(s):  
Zdeněk Knejzlík ◽  
Michal Doležal ◽  
Klára Herkommerová ◽  
Kamila Clarova ◽  
Martin Klima ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
De Novo ◽  
Gene Knockout ◽  
Guanosine Monophosphate ◽  
Inosine Monophosphate Dehydrogenase ◽  
Purine Salvage ◽  
Inosine Monophosphate ◽  
Salvage Pathways ◽  
Cbs Domains ◽  
Purine Salvage Pathways

Purine metabolism plays a pivotal role in bacterial life cycle, however, regulation of the de novo and purine salvage pathways have not been extensively detailed in mycobacteria. By gene knockout, biochemical and structural analyses, we identified Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) guaB1 gene product as a novel type of guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine salvage pathway and contains cystathione β-synthase (CBS) domains with atypical orientation in the octamer. CBS domains share a much larger interacting area with a conserved catalytic domain in comparison with the only known CBS containing protozoan GMPR and closely related inosine monophosphate dehydrogenase structures. Our results revealed essential effect of pH on allosteric regulation of Msm GMPR activity and oligomerization with adenine and guanosine nucleotides binding to CBS domains.Bioinformatic analysis indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

scholarly journals GuaA and GuaB Are Essential for Borrelia burgdorferi Survival in the Tick-Mouse Infection Cycle

2009 ◽  
Vol 191 (20) ◽  
pp. 6231-6241 ◽  
Author(s):  
Mollie W. Jewett ◽  
Kevin A. Lawrence ◽  
Aaron Bestor ◽  
Rebecca Byram ◽  
Frank Gherardini ◽  
...  
Keyword(s):  
De Novo ◽  
Infection Cycle ◽  
Purine Salvage ◽  
Dna And Rna ◽  
De Novo Purine Biosynthesis ◽  
Mouse Infection ◽  
Number Of Genes ◽  
Salvage Pathways ◽  
Purine Salvage Pathways ◽  
Enzymatic Pathways

ABSTRACT Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of B orrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.

scholarly journals Effect of hypoxia on purine metabolism of human skeletal muscle cells

Biotecnia ◽  
2021 ◽  
Vol 23 (2) ◽  
Author(s):  
Tania Zenteno-Savín ◽  
Crisalejandra Rivera-Pérez ◽  
Ramón Gaxiola-Robles ◽  
Norma Olguín-Monroy ◽  
Orlando Lugo-Lugo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Muscle Cells ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Purine Metabolism ◽  
Inosine Monophosphate Dehydrogenase ◽  
Inosine Monophosphate ◽  
Hypoxic Conditions ◽  
Human Skeletal Muscle Cells

Mammals experience some degree of hypoxia during their lifetime. In response to hypoxic challenge, mammalian cells orchestrate specific responses at transcriptional and posttranslational level which lead to changes in the purine metabolites in order to cope with threatening conditions. The aim of this study was to evaluate the response of the enzymes involved in the purine metabolism of human muscle cells to hypoxic conditions. Muscle cells in culture were exposed to hypoxia and the enzymatic activity of inosine monophosphate dehydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) as well as their transcript expression were quantified under normoxic and hypoxic conditions. Purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine diphosphate (GDP) and guanosine triphosphate (GTP)) concentrations were also quantified. Significant reduction of IMPDH activity and HX and IMP concentrations (p < 0.05) were observed after hypoxia, suggesting a decrease of de novo synthesis of purines. After hypoxia a global reduction of transcripts was observed, suggesting a reduction of the metabolic machinery of purine metabolism to new steady states that balance ATP demand and ATP supply pathways.

Regulation of the purine salvage pathway in rat liver

1992 ◽  
Vol 262 (3) ◽  
pp. E344-E352 ◽  
Author(s):  
Y. A. Kim ◽  
M. T. King ◽  
W. E. Teague ◽  
G. A. Rufo ◽  
R. L. Veech ◽  
...  
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Equilibrium Constants ◽  
Purine Metabolism ◽  
Purine Salvage ◽  
Rate Limiting Step ◽  
Delta G ◽  
Salvage Pathways ◽  
Rate Limiting

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

scholarly journals APRT SELECTION SYSTEM IN PLANTS

HortScience ◽  
1992 ◽  
Vol 27 (11) ◽  
pp. 1160f-1160
Author(s):  
Sunita K. Agarwal ◽  
David J. Schultz ◽  
Dennis A. Schaff
Keyword(s):  
De Novo ◽  
Selection System ◽  
Wild Type ◽  
Purine Salvage ◽  
Toxic Plants ◽  
Adenine Analogues ◽  
Salvage Pathways ◽  
Cloned Gene ◽  
High Degree ◽  
Adenine Derivatives

Most cells have an active turnover of many of their nucleic acids (particularly some types of RNA) which through degradative processes result in the release of adenine, guanine and hypoxanthine. These free purines are converted to their corresponding nucleotides through salvage pathways. Adenine is converted to its nucleotide form AMP by Adenine phosphoribosyltransferase (APRT) which is one of the enzymes associated with the purine salvage pathway. Since all organisms have a de novo pathway for the formation of AMP, APRT is classified as a `salvage enzyme'. The APRT enzyme, in general, does not show a high degree of specificity for the exact structure of adenine and can also act on cytokinins and adenine derivatives like 2,6-diaminopurine, 2-fluoroadenine and 6-methylpurine. The APRT enzyme can utilize adenine analogues as substrate and convert them into their nucleotide forms which are toxic. Plants that lack APRT activity (APRT-plants) survive in the presense of these analogues. The amount of adenine analogue used for selecting APRT-plants is such that it kills all APRT+ (wild type) plants. APRT+ plants survive when grown in the presense of azaserine and alanosine that block de novo synthesis of AMP. APRT-plants transformed with the wild type cloned gene can be selected from a mixture of transformed and non-transformed plants by selecting in the presense of adenine, azaserine and alanosine. The presense of APRT activity can be confirmed by assaying for the APRT enzyme. APRT activity has been detected in many plant species. The presense of a positive forward and backward selection system can thus allow the use of APRT as a selectable marker in plant gene transfer systems.

scholarly journals Purine Salvage Pathways among Borrelia Species

2007 ◽  
Vol 75 (8) ◽  
pp. 3877-3884 ◽  
Author(s):  
Jonas Pettersson ◽  
Merry E. Schrumpf ◽  
Sandra J. Raffel ◽  
Stephen F. Porcella ◽  
Cyril Guyard ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Relapsing Fever ◽  
Purine Salvage ◽  
Borrelia Hermsii ◽  
Reverse Transcription Pcr ◽  
Cell Densities ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Salvage Pathways ◽  
Purine Salvage Pathways

ABSTRACT Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.

A Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Overcomes Conventional Drug-Resistance in Multiple Mueloma Cells in the Bone Marrow Microenvironment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2468-2468
Author(s):  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Makoto Hamasaki ◽  
Raje Noopur ◽  
Kumar Shaji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
De Novo ◽  
Parp Cleavage ◽  
Inosine Monophosphate Dehydrogenase ◽  
Apoptotic Pathway ◽  
Inosine Monophosphate ◽  
Induced Apoptosis ◽  
Monophosphate Dehydrogenase

Abstract Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA) is a small molecule, selective, uncompetitive novel inhibitor directed against human IMPDH enzyme. IMPDH inhibitors have been demonstrated to induce growth arrest, and extensively investigated as immunosuppressants. Here we show that VX-944 inhibits growth of human multiple myeloma (MM) cell lines, including those resistant to conventional agents, via induction of apoptosis and S phase arrest in vitro. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs), do not protect against VX-944-induced MM cell growth inhibition. We next delineated the molecular mechanism of VX-944-induced MM cell death in the MM.1S human MM cell line. VX-944 induced apoptosis in MM.1S cells, confirmed by PARP cleavage as well as flow cytometric detection of the mitochondrial membrane protein 7A6 and TdT-mediated dUTP nick-end labelling (TUNEL) positive cells, without significant cleavage of caspases 3, 8 and 9. While the pan-caspase inhibitor z-VAD-fmk did not inhibit the VX-944-induced apoptosis and cell death suggesting that VX-944 triggers apoptosis in MM1.S cells primarily via caspase-independent pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin, melphalan and bortezomib, all of which activate caspases in MM cells and induce apoptosis, even in the presence of BMSCs. Taken together, our data demonstrate non-caspase-dependent apoptotic pathway triggered by VX-944 thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional and/or novel agents which trigger caspase activation. Our ongoing studies are delineating the mechanisms whereby VX-944 induces MM cell apoptosis.

scholarly journals Crystal structure of Schistosoma mansoni HGPRT isoform 1

2014 ◽  
Vol 70 (a1) ◽  
pp. C833-C833
Author(s):  
Larissa Romanello ◽  
Juliana de Souza ◽  
Louise Bird ◽  
Joanne Nettleship ◽  
Raymond Owens ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
De Novo ◽  
Structural Information ◽  
Guanosine Monophosphate ◽  
New Drugs ◽  
X Rays ◽  
Salvage Pathway ◽  
Purine Salvage ◽  
Space Groups ◽  
Key Enzyme

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 207 million people worldwide [1], and does not have the purine "de novo" pathway, depending entirely on the purine salvage pathway to supply its demands on purines [2]. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in this pathway and the only validated enzyme target of the pathway [3]. HGPRT catalyzes the PRPP dependent conversion of hypoxanthine/guanine to inosine monophosphate or guanine to guanosine monophosphate. HGPRT1 gene was amplified, cloned, expressed and purified at the Oxford Protein Production Facility (OPPF-UK). Robotic crystallization trials were performed and SmHGPRT crystallized in several conditions of the Morpheus crystallization kit: A4, A8, A9, and C9. The crystals appear about a day and have about 30 μM in greatest dimension. About a hundred crystals were screened with x-rays on the macromolecular crystallography beamlines I02 and I24 at Diamond Light Source. 21 datasets was collected from 2.97 to 4.11Å resolution. A solution was obtained for HGPRT1 belongs space group P212121 in a dataset to 3.4Å resolution, with four monomer in the ASU. The structure was solved by the program Phaser using HGPRT human as a search model. The refinement is being carried out by program Phenix. The density map is acceptable for the resolution but a great manual work of interpretation is necessary for the refinement of this structure. The most important is the demonstration that it was possible to crystallize and collect data of SmHGPRT. A major effort will be undertaken to improve the size and diffraction power of HGPRT crystals as well as in the resolution of the structure of HGPRT in other space groups. This structure will increase the structural information available about the Schistosoma mansoni purine salvage pathway.

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