Active site localization in a viral mRNA capping enzyme.

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• Reovirus mRNA capping is essential for viral gene expression.
• This process is catalyzed by the virion structural polypeptide lambda 2, a guanylyltransferase.
• The enzyme forms a phosphoamide-linked enzyme-pG covalent complex as a reaction intermediate.

Purpose of the Study:

• To identify the nucleotide attachment site on the lambda 2 guanylyltransferase.
• To pinpoint the specific amino acid residue involved in the capping reaction intermediate.

Main Methods:

• Incubation of reovirus particles with [alpha-32P]GTP.
• Proteolytic cleavage of labeled virus particles.
• Analysis of fragments using sequence-directed antibodies.
• Alkali digestion, oxidation, and beta-elimination of the labeled intermediate.

Main Results:

• The nucleotide attachment site was localized to amino acids 213-269 of lambda 2.
• This region contains potential GMP acceptors: one lysine, one arginine, and four histidine residues.
• Phospholysine was identified as the sole phosphoamino acid in the intermediate, indicating lysine 226 as the active site.

Conclusions:

• The active site of the reovirus guanylyltransferase lambda 2 is localized to a region containing lysine 226.
• This lysine residue is covalently modified during the formation of the enzyme-pG intermediate.
• These findings elucidate the catalytic mechanism of reovirus mRNA capping.