The m7GpppN cap structure of eukaryotic
mRNA is formed cotranscriptionally by the sequential action of three
enzymes:
RNA triphosphatase,
RNA guanylyltransferase, and
RNA (guanine-7)-methyltransferase. A multifunctional
polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional
guanylyltransferase polypeptides that lack triphosphatase and
methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent
enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping
enzymes. We performed a structure-function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae
guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping
enzyme. The results also allowed us to identify the capping
enzyme of Caenorhabditis elegans. The 573-amino
acid nematode
protein consists of a C-
terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16
amino acids that are essential for Ceg1 function, and an N-terminal
phosphatase domain that bears no resemblance to the
vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of
protein phosphatases that act via a covalent phosphocysteine intermediate.