Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase.
The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure-function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal mutants that displayed residual activity in vitro. We discuss our findings in light of a model of the Michaelis complex derived from crystal structures of AdoHcy-bound vaccinia cap methyltransferase and GTP-bound cellular cap methyltransferase. The structure-function data yield a coherent picture of the vaccinia cap methyltransferase active site and the determinants of substrate specificity and affinity.
Zheng
Sushuang
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Shuman
Stewart
S
D019943:Amino Acid Substitution; D019281:Dimerization; D005814:Genes, Viral; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase