Poxvirus mRNA cap methyltransferase: Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery Journal Article

  

Authors:	Schwer, B.; Hausmann, S.; Schneider, S.; Shuman, S.
Article Title:	Poxvirus mRNA cap methyltransferase: Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery
Abstract:	The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540-844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1Δ cells. We performed a genetic screen for mutations in the catalytic subunit that bypassed the requirement for the stimulatory subunit in vivo. We thereby identified missense changes in vicinal residues Tyr-752 (to Ser, Cys, or His) and Asn-753 (to Ile), which are located in the cap guanine-binding pocket. Biochemical experiments illuminated a mechanism of intersubunit allostery, whereby the vD12 subunit enhances the affinity of the catalytic subunit for AdoMet and the cap guanine methyl acceptor by 6- and 14-fold, respectively, and increases kcat by a factor of 4. The bypass mutations elicited gains of function in both vD12-independent and vD12-dependent catalysis of cap methylation in vitro when compared with wild-type vD1-(540-844). These results highlight the power of yeast as a surrogate model for the genetic analysis of interacting poxvirus proteins and demonstrate that the activity of an RNA processing enzyme can be augmented through selection and protein engineering. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords:	controlled study; unclassified drug; missense mutation; mutation; nonhuman; proteins; genes; serine; protein binding; alleles; tyrosine; rna; methyltransferase; methyltransferases; kinetics; messenger rna; guanine; saccharomyces cerevisiae; rna, messenger; vaccinia virus; dimerization; isoleucine; catalysis; protein subunit; enzyme kinetics; molecular biology; genetic screening; catalytic domain; mutagenesis; cysteine; asparagine; histidine; virus protein; enzyme active site; mutations; allosterism; poxvirus; poxviridae; saccharomyces; allosteric site; protein engineering; poxvirus proteins; poxvirus subunits; protein vd1; protein vd12
Journal Title:	Journal of Biological Chemistry
Volume:	281
Issue:	28
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Date Published:	2006-07-14
Start Page:	18953
End Page:	18960
Language:	English
DOI:	10.1074/jbc.M602867200
PUBMED:	16707499
PROVIDER:	scopus
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-33745868309&partnerID=40&md5=a84a9ff8489000bdd68500a4a6cdf82e
Notes:	--- - "Cited By (since 1996): 9" - "Export Date: 4 June 2012" - "CODEN: JBCHA" - "Source: Scopus"

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