Mutational analysis of a mammalian reovirus mRNA capping enzyme.
The amino-terminal 42-kDa region of the 144-kDa mammalian reovirus lambda 2 protein is a guanylyltransferase. It catalyzes the transfer of GMP from GTP to the 5' end of 5' -diphosphorylated mRNA via a phosphoamide with Lys-190. This amino acid is located at the base of a deep cleft. Based on sequence comparisons, the Kx[V/L/I]S motif is present in all known and proposed guanylyltransferases of the family Reoviridae. The requirement for this conserved sequence and other regions of the enzyme was analyzed by site-directed mutagenesis. Based on the enzymatic activity of the mutants, Lys-190 and Asp-191 are the only amino acids of the (190)KDLS sequence that are necessary for enzymatic activity. Since Asp-191 has its side chain oriented away from the cleft, most likely it plays an indirect role in forming a functional guanylyltransferase.
Luongo
Cindy L
CL
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. cindy_luongo@microbio.uab.edu
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D012089:Mammalian orthoreovirus 3 / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012315:RNA Caps; D012087:Reoviridae / Q000201:enzymology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D012315:RNA Caps; D014758:Viral Core Proteins; D014764:Viral Proteins; C071342:reovirus protein lambda2; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D000409:Alanine