Mutational Analysis of a Mammalian Reovirus mRNA Capping Enzyme

• Published in: Biochemical and biophysical research communications 2002-03, Vol.291 (4), p.932-938
• Main Author: Luongo, Cindy L.
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Binding Sites; capping enzyme; Conserved Sequence; DNA Mutational Analysis; guanylyltransferase; l2 protein; Mammalian orthoreovirus 3 - enzymology; Molecular Sequence Data; mRNA; Mutagenesis, Site-Directed; nucleotidyl transfer; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - genetics; Nucleotidyltransferases - metabolism; orbivirus; Reoviridae; Reoviridae - enzymology; Reovirus; RNA capping; RNA Caps; rotavirus; Sequence Alignment; site-directed mutagenesis; Structure-Activity Relationship; Viral Core Proteins - chemistry; Viral Core Proteins - genetics; Viral Core Proteins - metabolism; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; virus
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2002.6520

The amino-terminal 42-kDa region of the 144-kDa mammalian reovirus λ2 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 190KDLS 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.