Conversion Of Guanosine Triphosphate Research Articles

Soluble Guanylate Cyclase Stimulators and Activators: Where are We and Where to Go?

Soluble Guanylate Cyclase (sGC) is the intracellular receptor of Nitric Oxide (NO). The activation of sGC results in the conversion of Guanosine Triphosphate (GTP) to the secondary messenger cyclic Guanosine Monophosphate (cGMP). cGMP modulates a series of downstream cascades through activating a variety of effectors, such as Phosphodiesterase (PDE), Protein Kinase G (PKG) and Cyclic Nucleotide-Gated Ion Channels (CNG). NO-sGC-cGMP pathway plays significant roles in various physiological processes, including platelet aggregation, smooth muscle relaxation and neurotransmitter delivery. With the approval of an sGC stimulator Riociguat for the treatment of Pulmonary Arterial Hypertension (PAH), the enthusiasm in the discovery of sGC modulators continues for broad clinical applications. Notably, through activating the NO-sGC-cGMP pathway, sGC stimulator and activator potentiate for the treatment of various diseases, such as PAH, Heart Failure (HF), Diabetic Nephropathy (DN), Systemic Sclerosis (SS), fibrosis as well as other diseases including Sickle Cell Disease (SCD) and Central Nervous System (CNS) disease. Here, we review the preclinical and clinical studies of sGC stimulator and activator in recent years and prospect for the development of sGC modulators in the near future.

The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ₀) Transport.

Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ0), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ0 to 7-aminomethyl-7-deazaguanine (preQ1). PreQ1 is inserted into tRNAs by tRNA guanine(34) transglycosylase (TGT). The inserted base preQ1 is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ0 synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ0, preQ1 or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ0/preQ1 transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ0 transport.

Mechanistic insights into nitric oxide activation of H‐NOX sensor proteins (1013.18)

Nitric oxide (NO) is a freely diffusible gas that is critical for normal cardiovascular function through the regulation of processes such as blood vessel homeostasis and inhibition of platelet aggregation. An integral component of mammalian NO signaling is soluble guanylate cyclase (sGC), the primary receptor for NO, whereby sensitive and selective binding of NO to its heme‐containing H‐NOX domain results in conversion of guanosine triphosphate (GTP) to the secondary messenger cyclic guanosine monophosphate (cGMP). The heme cofactor of sGC exists in a 5‐coordinate ferrous state (Fe(II)) with histidine as the proximal ligand. NO binding to the distal heme site yields a transient 6‐coordinate complex where, the trans effect exhibited by NO weakens the iron‐histidine bond resulting in histidine dissociation to yield an active, 5‐coordinate NO complex (Fe(II)‐NO). Several biochemical studies have determined that severing of the iron‐histidine bond is required for activation, however, the lack of a 5‐coordinate NO H‐NOX structure has limited molecular level insights into the NO activation mechanism. Toward this end, crystal structures of a bacterial H‐NOX protein free and complexed with NO have been determined and reveal a conformational change resulting from the breakage of the iron‐histidine bond. It is hypothesized that this conformational change is the mechanism by which H‐NOX proteins regulate their signaling effector partners.

Overexpression, purification, crystallization and preliminary X-ray analysis of putative molybdenum cofactor biosynthesis protein C (MoaC2) from Mycobacterium tuberculosis H37Rv.

Rv0864 (MoaC2) from Mycobacterium tuberculosis is one of the enzymes in the molybdenum cofactor (Moco) biosynthesis pathway. Together with MoaA, MoaC is involved in the conversion of guanosine triphosphate (GTP) to precursor Z, the first step in Moco synthesis. Full-length MoaC2 (17.5 kDa, 167 residues) was cloned in Escherichia coli and purified to homogeneity. Crystals of recombinant M. tuberculosis MoaC2 were grown by vapour diffusion using a hanging-drop setup. Diffracting crystals grew in a condition in which 3 µl protein solution at 10.5 mg ml(-1) was mixed with 1.5 µl reservoir solution (0.025 M potassium sodium tartrate tetrahydrate pH 8.0) and equilibrated against 1000 µl reservoir solution. Diffraction data extending to 2.5 Å resolution were collected at 100 K. The crystal belonged to the cubic space group P2(1)3, with unit-cell parameter 94.5 Å. Matthews coefficient (V(M)) calculations suggested the presence of two molecules in the asymmetric unit, corresponding to a solvent content of about 39%. Molecular-replacement calculations using the E. coli homologue as the search model gave an unambiguous solution.

Plasmodium falciparum: Effect of antimalarial drugs, malaria pigment (β-haematin) and Plasmodium falciparum lysate on monocyte GTP-cyclohydrolase 1 gene expression

In interferon-γ activated human macrophages, GTP-cyclohydrolase 1 catalyses the conversion of guanosine triphosphate to 7,8-dihydroneopterin triphosphate, which is dephosphorylated and oxidized to form neopterin. Elevated levels of neopterin have been detected in the urine and serum of malaria-infected patients. In this study, U937 cells were treated with interferon-γ and one of the following antimalarial drugs: amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine or quinine. The effects of treating the U937 cells with malaria pigment (β-haematin), latex beads, or Plasmodium falciparum-infected-red blood cell lysates were also investigated. U937 GTP-cyclohydrolase 1 mRNA expression was monitored using reverse-transcriptase-quantitative PCR. Artemisinin, primaquine, and quinine down-regulated GTP-cyclohydrolase 1 gene expression 1.26-, 1.29-, and 1.63-fold, respectively. The remaining drugs had insignificant effects. β-haematin up-regulated GTP-cyclohydrolase 1 mRNA expression 1.18-fold, whereas P. falciparum-infected red blood cell lysate down-regulated expression 1.56-fold. These results show the differing immunomodulatory actions of antimalarial drugs and malaria pigment taking place in monocytes.

Magnesium cation initiates nucleoside triphosphate to monophosphate conversion by a radical mechanism: Quantum molecular dynamics simulation

A DFT:B3LYP (6-311G** basis set) quantum molecular dynamics simulation was used to study the mechanism of Mg2+-induced conversion of guanosine triphosphate (GTP) to guanosine monophosphate (GMP). The computations were performed at 310 K in a bath of 178 water molecules surrounding the Mg(H2O)2-GTP complex and mimicking the hydration shells. Dissociation of the Mg2+-GTP complex (process duration, 5 ps) produces two phosphate anions (Pi), a hydrated Mg2+ cation, atomic oxygen, and a highly reactive GMP radical. This radical appears as a result of the action of Mg2+, which initiates the radical mechanism of GTP cleavage. At the initial stage of the interaction with GTP, Mg2+ is reduced to Mg+, producing an ion-radical pair +Mg·-·GTP3−. In the absence of Mg2+, an inert GMP molecule forms instead of the GMP radical as a result of hydrolytic cleavage of GTP via an ionic mechanism. Presumably, formation of the GMP radical and analogous radicals with adenosine, cytidine, thymidine, and uridine is a key point in the syntheses of deoxyribonucleic and ribonucleic acids.

GTPase activating proteins: structural and functional insights 18 years after discovery.

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (P(i)) by guanine nucleotide binding proteins (GNBPs) is a fundamental process in living cells and represents an important timer in intracellular signalling and transport processes. While the rate of GNBP-mediated GTP hydrolysis is intrinsically slow, direct interaction with GTPase activating proteins (GAPs) accelerates the reaction by up to five orders of magnitude in vitro. Eighteen years after the discovery of the first GAP, biochemical and structural research has been accumulating evidence that GAPs employ a much wider spectrum of chemical mechanisms than had originally been assumed, in order to regulate the chemical players on the catalytic protein-protein interaction stage.

Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by guanine nucleotide-binding proteins (GNBPs) is a fundamental enzyme reaction in living cells that acts as an important timer in a variety of biological processes. This reaction is intrinsically slow but can be stimulated by GTPase-activating proteins (GAPs) by several orders of magnitude. In the present study, we synthesized and characterized a new fluorescent nucleotide, 2'(3')-O-(N-ethylcarbamoyl-(5''-carboxytetramethylrhodamine) amide)-GTP, or tamraGTP, which is sensitive towards conformational changes of certain GNBPs induced by GTP hydrolysis. Unlike other fluorescent nucleotides, tamra-GTP allows real-time monitoring of the kinetics of the intrinsic and GAP-catalyzed GTP hydrolysis reactions of small GNBPs from the Rho family.

6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human cells using high-performance liquid chromatography with fluorescence detection of biopterin

An assay for 6-pyruvoyl tetrahydropterin synthase, the second enzyme in the conversion of guanosine triphosphate into tetrahydrobiopterin, has been developed. Cell extracts were incubated with enzymatically prepared dihydroneopterin triphosphate (80 μ M) in the presence of Mg 2+ (12 m M), excess sepiapterin reductase (EC 1.1.1.153) (2 nmol/min) and NADPH (2 m M). 6-Pyruvoyl tetrahydropterin, the product of the reaction, was thus converted into tetrahydrobiopterin. After oxidation of the reduced biopterin derivatives in acidic iodine solution, biopterin was enriched and separated from the abundant neopterin phosphates by solid-phase extraction on a strong cation exchanger. Biopterin was then directly eluted on a reversed-phase liquid chromatographic column and detected fluorimetrically using excitation at 353 nm and emission at 438 nm. The biopterin concentrations formed by the coupled enzyme reaction increased linearly with incubation times up to 90 min. The assay allows the quantification of 6-pyruvoyl tetrahydropterin synthase in cultured human cells.

Angiotensin II stimulates guanylate cyclase activity in aorta, heart, and kidney.

The objective of the present investigation was to determine whether angiotensin II at physiological levels has part of its mechanism of action through stimulation of the activity of guanylate cyclase (EC 4.6.1.2), the enzyme that catalyzes the conversion of guanosine triphosphate to cyclic GMP. Angiotensin II enhanced guanylate cyclase activity three-to fivefold in rat aorta, heart, and kidney at a concentration of 1 nM. Dose-response curves revealed that near maximal stimulation of guanylate cyclase with angiotensin II was observed at a concentration as low as 10 pM. The guanylate cyclase cofactor manganese was necessary for the maximal enhancement of guanylate cyclase by angiotensin II. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of angiotensin II at the cellular level.