Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism Journal Article
| Authors: | Martins, A.; Shuman, S. |
| Article Title: | Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism |
| Abstract: | The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a 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 β barrel (the so-called 'triphosphate tunnel'). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of γ phosphate hydrolysis. |
| Keywords: | controlled study; unclassified drug; gene cluster; mutation; review; nonhuman; protein analysis; amino acid substitution; phosphatase; enzyme activation; enzyme activity; acid anhydride hydrolases; structure activity relation; mutational analysis; amino acid sequence; molecular sequence data; sequence homology, amino acid; kinetics; messenger rna; dna viruses; vaccinia virus; binding sites; alanine; catalysis; protein family; glutamic acid; virus strain; protozoa; fungi; lysine; arginine; metal; chlorella virus; viral proteins; enzyme active site; enzyme conformation; peptide mapping; magnesium; poxvirus; poxviridae; rna capping; orthology; nucleotidyltransferases; manganese; baculovirus; baculoviridae; unidentified baculovirus; priority journal; acid protein; basic protein; protein lef4 |
| Journal Title: | Nucleic Acids Research |
| Volume: | 31 |
| Issue: | 5 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2003-03-01 |
| Start Page: | 1455 |
| End Page: | 1463 |
| Language: | English |
| DOI: | 10.1093/nar/gkg244 |
| PUBMED: | 12595553 |
| PROVIDER: | scopus |
| PMCID: | PMC149837 |
| DOI/URL: | |
| Notes: | Export Date: 25 September 2014 -- Source: Scopus |
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