Encephalitozoon cuniculi mRNA Cap (guanine N-7) methyltransferase: Methyl acceptor specificity, inhibition by S-adenosylmethionine analogs, and structure-guided mutational analysis Journal Article

  

Authors:	Hausmann, S.; Zheng, S.; Fabrega, C.; Schneller, S. W.; Lima, C. D.; Shuman, S.
Article Title:	Encephalitozoon cuniculi mRNA Cap (guanine N-7) methyltransferase: Methyl acceptor specificity, inhibition by S-adenosylmethionine analogs, and structure-guided mutational analysis
Abstract:	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 7GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K m 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 μM AdoMet is inhibited by the reaction product AdoHcy (IC 50 4 μ) and by substrate analogs sinefungin (IC 50 1.5 μM), aza-AdoMet (IC 50 100 μM), and carbocyclic aza-AdoMet (IC 50 35 μM). 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords:	unclassified drug; methylation; mutation; mutation, missense; nonhuman; binding affinity; animals; complex formation; enzyme inhibition; amino acid substitution; ph; in vivo study; enzyme activity; structure-activity relationship; rna; methyltransferase; methyltransferases; messenger rna; guanine; adenosine; enzyme inhibitors; s-adenosylhomocysteine; recombinant proteins; substrate specificity; binding site; crystal structure; models, molecular; binding sites; molecular structure; alanine; catalysis; adenosine triphosphate; crystallization; ic 50; enzyme specificity; biochemistry; guanosine triphosphate; aspartic acid; mutagenesis; enzyme purification; dinucleotide; enzymes; lysine; hydrogen-ion concentration; monomer; enzyme mechanism; phenylalanine; mrna; functional group; s-adenosylmethionine; rna methyltransferase; capped rna; s adenosylmethionine; methyl group; microorganisms; cytidine triphosphate; sinefungin; substitution reactions; adenosinetriphosphate; encephalitozoon cuniculi; uridine triphosphate; protein ecm1
Journal Title:	Journal of Biological Chemistry
Volume:	280
Issue:	21
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Date Published:	2005-05-27
Start Page:	20404
End Page:	20412
Language:	English
DOI:	10.1074/jbc.M501073200
PUBMED:	15760890
PROVIDER:	scopus
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-20144383545&partnerID=40&md5=9b3f640b6ec73356c8efbc1680794924
Notes:	--- - "Cited By (since 1996): 13" - "Export Date: 24 October 2012" - "CODEN: JBCHA" - "Source: Scopus"

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