guanosine-triphosphate and 5-6-7-8-tetrahydrofolic-acid

Folate pathway is a key target for the development of new drugs against infectious diseases since the discovery of sulfa drugs and trimethoprim. The knowledge about this pathway has increased in the last years and the catalytic mechanism and structures of all enzymes of the pathway are fairly understood. In addition, differences among enzymes from prokaryotes and eukaryotes could be used for the design of specific inhibitors. In this review, we show a panorama of progress that has been achieved within the folate pathway obtained in the last years. We explored the structure and mechanism of enzymes, several genetic features, strategies, and approaches used in the design of new inhibitors that have been used as targets in pathogen chemotherapy.

Topics: Animals; Anti-Infective Agents; Bacteria; Bacterial Infections; Biosynthetic Pathways; Communicable Diseases; Drug Design; Folic Acid; Folic Acid Antagonists; Fungi; Guanosine Triphosphate; Humans; Models, Molecular; Mycoses; Tetrahydrofolates

In Bacillus subtilis a sophisticated regulatory circuit that involves Z nucleoside triphosphate (ZTP) is recruited to optimize cellular zinc distribution when cytoplasmic zinc is scarce. This process uses enzymatic reactions to measure the pool of available zinc ions and amplifies this signal to control the activity of zinc chaperones. The ZTP-dependent regulatory circuit that is exploited for zinc homeostasis controls purine and folate biosynthesis, which starts with GTP as initial substrate. Low concentrations of formyl-tetrahydrofolate (fTHF) lead to accumulation of the intermediate 5'-phosphoribosyl-4-carboxyamide-5-aminoimidazole (AICAR or ZMP), which is pyrophosphorylated by another intermediate to ZTP. This alarmone activates expression of genes using a ZTP-dependent riboswitch in many bacterial strains. In this way, the cellular folate concentration controls folate biosynthesis via the enzymatic activity of the fTHF-dependent AICAR-transforming reaction. Zinc distribution control is layered onto this circuit. The 'sensor' is the activity of the initial reaction of folate synthesis from GTP, which is catalyzed by a zinc-dependent enzyme FolE

Topics: Aminoimidazole Carboxamide; Bacillus subtilis; Bacterial Proteins; Guanosine Triphosphate; Homeostasis; Molecular Chaperones; Ribonucleotides; Tetrahydrofolates; Zinc

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH(4)), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G(+)) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins includes the 6-pyruvoyltetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologues, all members of the COG0720 family that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H(2)NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH(4) pathway, PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway, and PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, phylogenetic codistribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis and resulted in the identification of PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families extensive comparative genomic analysis beyond homology is required to correctly predict function.

Topics: Amino Acid Motifs; Archaeal Proteins; Biopterins; Genetic Complementation Test; Guanosine; Guanosine Triphosphate; Kinetics; Models, Molecular; Molecular Sequence Data; Neopterin; Nucleoside Q; Phosphorus-Oxygen Lyases; Phylogeny; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Sulfolobus; Tetrahydrofolates

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Structure, Secondary; Protein Structure, Tertiary; RNA, Transfer; Sequence Alignment; Tetrahydrofolates; Uridine

Intracerebral folate injections produce convulsions and brain lesions, folic acid itself and tetrahydrofolate being more potent toxins than 5-methyltetrahydrofolate, the primary folate of mammalian extracellular fluids. Folates are known to excite neurons, by unknown mechanisms Folates stimulate GTP binding and GTPase activity in slime molds. We observed folate stimulation of GTP gamma S binding and inhibition of high affinity GTPase activity in rat brain membranes. Three fold stimulation of GTP gamma S binding was observed in cerebellar membranes treated with 50 microM FA. Folic acid (FA), dihydrofolate (DHF) and tetrahydrofolate (THF) were much more potent than 5-methyltetrahydrofolate in this regard. The effect varies between brain regions and was greatest in cerebellar and hippocampal membranes. Folates inhibit GTPase activity, with DHF and FA being the most potent and maximum inhibition being to 33% of control values. We find high affinity guanine nucleotide sensitive binding of [3H]FA in cerebellar membranes, another response typical of G protein coupled membrane receptors. Folates were also shown to stimulate the release of [3H]GDP from brain membranes. These effects are seen in washed brain membranes and can not be explained by any known folate metabolic or coenzyme functions. They resemble the effects of cholera toxin, except for their reversibility. They may be relevant to known folate neuroexcitant effects of folates.

Topics: Animals; Brain; Cell Membrane; Cerebellum; Corpus Striatum; Folic Acid; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hippocampus; Male; Rats; Rats, Inbred Strains; Tetrahydrofolates; Thionucleotides