The
RNA triphosphatase component of vaccinia virus
mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of
metal-dependent
phosphohydrolases that includes the
RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two
glutamate-containing motifs (A and C) that form the
metal-binding site. Most of the family members resemble the fungal and Chlorella virus
enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded beta barrel (the so-called "
triphosphate tunnel"). Here we queried whether
vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of
amino acids required for
vaccinia triphosphatase activity. We identified four new essential side chains in
vaccinia D1 via
alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three
basic amino acids that likely compose the
vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus
RNA capping
enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel
proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the
metal-dependent
RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping
enzyme, recommends the poxvirus
RNA triphosphatase as a molecular target for antipoxviral
drug discovery.