Poxvirus mRNA cap methyltransferase. Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery.
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 abd1delta 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.
Schwer
Beate
B
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Hausmann
Stéphane
S
Schneider
Susanne
S
Shuman
Stewart
S
D000483:Alleles; D000495:Allosteric Site; D020134:Catalytic Domain; D019281:Dimerization; D006147:Guanine / Q000737:chemistry; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics* / Q000502:physiology*; D009154:Mutation; D011212:Poxviridae / Q000201:enzymology*; D011485:Protein Binding; D015202:Protein Engineering; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics
D012333:RNA, Messenger; D006147:Guanine; D012313:RNA; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase