Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site Journal Article

   

Authors:	Zheng, S.; Shuman, S.
Article Title:	Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site
Abstract:	RNA guanine-N7 methyltransferase catalyzes the third step of eukaryal mRNA capping, the transfer of a methyl group from AdoMet to GpppRNA to form m 7GpppRNA. Mutational and crystallographic analyses of cellular and poxvirus cap methyltransferases have yielded a coherent picture of a conserved active site and determinants of substrate specificity. Models of the Michaelis complex suggest a direct in-line mechanism of methyl transfer. Because no protein contacts to the guanine-N7 nucleophile, the AdoMet methyl carbon (Cε) or the AdoHcy sulfur (Sδ) leaving group were observed in ligand-bound structures of cellular cap methyltransferase, it was initially thought that the enzyme facilitates catalysis by optimizing proximity and geometry of the donor and acceptor. However, the structure of AdoHcy-bound vaccinia virus cap methyltransferase revealed the presence of an N-terminal "lid peptide" that closes over the active site and makes multiple contacts with the substrates, including the AdoMet sulfonium. This segment is disordered in the vaccinia apoenzyme and is not visible in the available structures of cellular cap methyltransferase. Here, we conducted a mutational analysis of the vaccinia virus lid peptide ( 545DKFRLNPEVSYFTNKRTRG563) entailing in vivo and in vitro readouts of the effects of alanine and conservative substitutions. We thereby identified essential functional groups that interact with the AdoMet sulfonium (Tyr555, Phe556), the AdoMet adenine (Asn550), and the cap triphosphate bridge (Arg560, Arg562). The results suggest that van der Waals contacts of Tyr555 and Phe556 to the AdoMet Sδ and Cε atoms, and the electron-rich environment around the sulfonium, serve to stabilize the transition state of the transmethylation reaction. Copyright © 2008 RNA Society.
Keywords:	controlled study; gene mutation; methylation; nonhuman; amino acid substitution; enzyme activity; structure activity relation; structure-activity relationship; tyrosine; mutational analysis; methyltransferase; methyltransferases; amino acid sequence; conserved sequence; molecular sequence data; amino terminal sequence; messenger rna; eukaryota; peptides; vaccinia virus; crystal structure; binding sites; dna mutational analysis; alanine; catalysis; multienzyme complexes; enzyme specificity; enzyme structure; phosphoric monoester hydrolases; ligand binding; phenylalanine; mrna capping; adenine; poxvirus; poxviridae; s-adenosylmethionine; rna capping; nucleotidyltransferases; virus enzyme; sulfonium; guanine 7 oxide; sulfonium derivative
Journal Title:	RNA
Volume:	14
Issue:	11
ISSN:	1355-8382
Publisher:	Cold Spring Harbor Laboratory Press
Date Published:	2008-11-01
Start Page:	2297
End Page:	2304
Language:	English
DOI:	10.1261/rna.1201308
PUBMED:	18799596
PROVIDER:	scopus
PMCID:	PMC2578867
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-55549130291&partnerID=40&md5=d40d545000569627365c15b2eb67fdc2
Notes:	--- - "Cited By (since 1996): 1" - "Export Date: 17 November 2011" - "CODEN: RNARF" - "Source: Scopus"

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