guanosine-triphosphate and sinefungin

The mRNA-capping process starts with the conversion of a 5'-triphosphate end into a 5'-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5'-5' phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2'OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2'O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, (N7Me)GpppG, and (N7Me)GpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2'O-methyltransferase activities.

Topics: Adenosine; Amino Acid Sequence; Binding Sites; Biological Assay; Crystallography, X-Ray; Flavivirus; Guanosine Triphosphate; Methyltransferases; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Structure, Secondary; Protein Structure, Tertiary; RNA Caps; S-Adenosylmethionine; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Viral Nonstructural Proteins

Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity at its N-terminal amino acid sequence and is responsible for formation of a type 1 cap structure, m(7)GpppAm(2'-O) in the viral genomic RNA. Optimal in vitro conditions for DENV2 2'-O-MTase activity were characterized using purified recombinant protein and a short biotinylated GTP-capped RNA template. Steady-state kinetics parameters derived from initial velocities were used to establish a robust scintillation proximity assay for compound testing. Pre-incubation studies showed that MTase-AdoMet and MTase-RNA complexes were equally catalytically competent and the enzyme supports a random bi bi kinetic mechanism. The assay was validated with competitive inhibitory agents, S-adenosyl-homocysteine and two homologues, sinefungin and dehydrosinefungin. A GTP-binding pocket present at the N-terminal of DENV2 MTase was previously postulated to be the cap-binding site. Interestingly, inhibition of the enzyme by GTP was two-fold lower than with RNA cap analogues, G[5']ppp[5']A and m(7)G[5']ppp[5']A and about three-fold poorer than a two-way methylated analogue, m(7)G[5']ppp[5']m(7)G. This assay allows rapid and highly sensitive detection of 2'-O-MTase activity and can be readily adapted for high-throughput screening for inhibitory compounds. It is suitable for determination of enzymatic activities of a wide variety of RNA capping MTases.

Topics: Adenosine; Binding Sites; Biochemistry; Dengue Virus; Enzyme Inhibitors; Guanosine Triphosphate; Kinetics; Methylation; Methyltransferases; S-Adenosylhomocysteine; Scintillation Counting; Sensitivity and Specificity; Viral Proteins

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m(7)GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K 0.1 mm) is higher than that for GTP (K(m) 1 mm) or GDP (K(m) 2.4 mm). Methylation of GTP by Ecm1 in the presence of 5 microm AdoMet is inhibited by the reaction product AdoHcy (IC(50) 4 microm) and by substrate analogs sinefungin (IC(50) 1.5 microm), aza-AdoMet (IC(50) 100 microm), and carbocyclic aza-AdoMet (IC(50) 35 microm). The crystal structure of an Ecm1.aza-AdoMet binary complex reveals that the inhibitor occupies the same site as AdoMet. Structure-function analysis of Ecm1 by alanine scanning and conservative substitutions identified functional groups necessary for methyltransferase activity in vivo. Amino acids Lys-54, Asp-70, Asp-78, and Asp-94, which comprise the AdoMet-binding site, and Phe-141, which contacts the cap guanosine, are essential for cap methyltransferase activity in vitro.

Topics: Adenosine; Animals; Binding Sites; Crystallization; Encephalitozoon cuniculi; Enzyme Inhibitors; Guanosine Triphosphate; Hydrogen-Ion Concentration; Methylation; Methyltransferases; Models, Molecular; Molecular Structure; Mutagenesis; Mutation, Missense; Recombinant Proteins; S-Adenosylhomocysteine; S-Adenosylmethionine; Structure-Activity Relationship; Substrate Specificity