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PDBsum entry 1z3c
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References listed in PDB file
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Key reference
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Title
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Encephalitozoon cuniculi mRNA cap (guanine n-7) methyltransferase: methyl acceptor specificity, Inhibition by s-Adenosylmethionine analogs, And structure-Guided mutational analysis.
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Authors
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S.Hausmann,
S.Zheng,
C.Fabrega,
S.W.Schneller,
C.D.Lima,
S.Shuman.
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Ref.
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J Biol Chem, 2005,
280,
20404-20412.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 4.
FIG. 4. Chemical structures of AdoMet, AdoHcy, and analogs
sinefungin, aza-AdoMet, and carbocyclic aza-AdoMet.
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Figure 8.
FIG. 8. Structures of Ecm1 and the active site. A, stereo
view of the structure of Ecm1 in complex with aza-AdoMet. B, a
2.2-Å simulated annealing omit map is shown contoured at
1.0  covering the aza-AdoMet
ligand. C, stereo view of the Ecm1 structure (Protein Data Bank
accession code 1RI2 [PDB]
) in complex with the GTP portion of the m7GpppG cap analog.
Potential hydrogen-bonding interactions are shown by dashed
lines; waters are rendered as red spheres. Amino acid residues
are labeled and shown in stick representation. aza-AdoMet and
GTP are shown in stick representation. The figure was generated
with Pymol software (W. L. DeLano, www.pymol.org).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
20404-20412)
copyright 2005.
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