Structure-function analysis of the mRNA cap methyltransferase of Saccharomyces cerevisiae Journal Article


Authors: Wang, S. P.; Shuman, S.
Article Title: Structure-function analysis of the mRNA cap methyltransferase of Saccharomyces cerevisiae
Abstract: The Saccharomyces cerevisiae mRNA cap methylating enzyme is a 436-amino acid protein encoded by the essential ABD1 gene. To identify structural features of ABD1 required for enzyme function, we introduced alanine mutations at 19 positions within a 205-amino acid region of similarity to the methyltransferase domain of the vaccinia capping enzyme. Three new recessive lethal mutations, E170A, D194A, and R206A, were identified. Structure- function relationships were clarified by introducing conservative substitutions at Glu-170, Asp-194, and Arg-206, and at Tyr-254 (an essential residue identified previously). Alleles E170D and D194E were viable, whereas E170Q and D194N were lethal; hence, acidic side chains were critical at both positions. R206K was viable, suggesting that a basic residue sufficed. Y254S was lethal, whereas Y254F was viable, albeit slow growing; thus, an aromatic side chain was important. The ABD1 mutations that were deleterious in vivo elicited catalytic defects in vitro. By studying the effects of amino- and carboxyl-terminal deletions, we defined a fully active catalytic domain of ABD1 from residues 130 to 426. Residues 110-129 were dispensable for methyltransferase activity in vitro, but essential for function in vivo. This analysis allowed us to delineate a subfamily of ABD1-like proteins within the superfamily of AdoMet-dependent methyltransferases. In addition, we identify a candidate Caenorhabditis elegans gene encoding a putative cap methyltransferase. All residues essential for ABD1 activity are conserved in the C. elegans homologue.
Keywords: gene mutation; sequence deletion; nonhuman; polymerase chain reaction; animals; allele; gene; amino acid substitution; carboxy terminal sequence; protein; enzyme activity; structure activity relation; tyrosine; blotting, western; methyltransferase; methyltransferases; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; messenger rna; saccharomyces cerevisiae; sequence alignment; recombinant proteins; immunoblotting; plasmids; amino acid; mutagenesis, site-directed; caenorhabditis elegans; alanine; catalysis; point mutation; aspartic acid; arginine; polyacrylamide gel electrophoresis; enzyme; vaccinia; viruses; genes, fungal; priority journal; article
Journal Title: Journal of Biological Chemistry
Volume: 272
Issue: 23
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1997-06-06
Start Page: 14683
End Page: 14689
Language: English
DOI: 10.1074/jbc.272.23.14683
PUBMED: 9169431
PROVIDER: scopus
DOI/URL:
Notes: Article -- Export Date: 17 March 2017 -- Source: Scopus
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  1. Shuang-Ping Wang
    4 Wang
  2. Stewart H Shuman
    547 Shuman