Abstract
Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.
Highlights
Vaccinia virus RNA capping enzyme, a heterodimer The intermediate (EpG) consists of a GMP residue linked of 95- and 31-kDa subunits, catalyzes transfer of GMP covalently to the95-kDa subunit via a phosphoamide bond to from GTP to the 5”diphosphate terminus of RNA via the €-aminogroup of a lysine [4,5,6]
We find that conservative amino acid substitutions at three outof four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation
The guanylyltransferase domain has been localized within the first 614 amino acids of the D l polypeptide based on the finding that a carboxyl-truncatedderivative of the large subsequences of capping enzymes and polynucleotide li- unit is activewhen expressed in gases from diverse sources suggests that KX(D/N)G bacteria[13]
Summary
A KTDGMOTIFESSENTIAL FOR ENZYME-GMPCOMPLEX FORMATION BY mRNA CAPPING ENZYME IS CONSERVED A T THE ACTIVE SITES OF RNAANDDNA LIGASES*. The guanylyltransferase domain (and the active site lysine) has been localized within the first 614 amino acids of the D l polypeptide based on the finding that a carboxyl-truncatedderivative of the large subsequences of capping enzymes and polynucleotide li- unit is active (in protein-GMP formation)when expressed in gases from diverse sources suggests that KX(D/N)G bacteria[13]. The conserved lysine is located at position 260 of the vaccinia D l polypeptide, i.e. within the guanylyltrans- These three steps are catalyzed by the RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase components of a capping enzyme complex containing 95- and 31-kDa virus-encoded subunits [2, 3].
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