Mutational analysis of baculovirus capping enzyme Lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity Journal Article
| Authors: | Martins, A.; Shuman, S. |
| Article Title: | Mutational analysis of baculovirus capping enzyme Lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity |
| Abstract: | The 464-amino acid baculovirus Lef4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The hydrolysis of 5′-triphosphate RNA and free NTPs by Lef4 is dependent on a divalent cation cofactor. RNA triphosphatase activity is optimal at pH 7.5 with either magnesium or manganese, yet NTP hydrolysis at neutral pH is activated only by manganese or cobalt. Here we show that Lef4 possesses an intrinsic magnesium-dependent ATPase with a distinctive alkaline pH optimum and a high Km for ATP (4 mM). Lef4 contains two conserved sequences, motif A (8IEKEISY14) and motif C (180LEYEF 184), which define the fungal/viral/protozoal family of metal-dependent RNA triphosphatases. We find by mutational analysis that Glu9, Glu11, Glu181, and Glu183 are essential for phosphohydrolase chemistry and likely comprise the metal-binding site of Lef4. Conservative mutations E9D and E183D abrogate the magnesium-dependent triphosphatase activities of Lef4 and transform it into a strictly manganese-dependent RNA triphosphatase. Limited proteolysis of Lef4 and ensuing COOH-terminal deletion analysis revealed that the NH 2-terminal 236-amino acid segment of Lef4 constitutes an autonomous triphosphatase catalytic domain. |
| Keywords: | unclassified drug; genetics; missense mutation; mutation; mutation, missense; nonhuman; molecular genetics; protein conformation; protein domain; metabolism; phosphatase; carboxy terminal sequence; protein degradation; ph; enzymology; rna triphosphatase; acid anhydride hydrolases; rna; chemistry; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; kinetics; messenger rna; enzyme analysis; dna viruses; nucleotide sequence; binding site; dna mutational analysis; amino acids; adenosine triphosphate; cobalt; sequence homology; structure analysis; enzyme specificity; adenosine triphosphatase; enzyme structure; hydrolysis; mutagenesis; molecular probe; enzymes; hydrogen-ion concentration; virus protein; viral proteins; divalent cation; cations, divalent; magnesium; rna capping; manganese; transferase; proteolysis; ph effects; microorganisms; baculovirus; virus enzyme; baculoviridae; molecular probes; hydrolase; positive ions; metal binding; adenosinetriphosphate; unidentified baculovirus; priority journal; article; support, u.s. gov't, p.h.s.; adenosinetriphosphatase; magnesium printing plates; lef4 protein; late expression factor 4, baculovirus |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 49 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-12-07 |
| Start Page: | 45522 |
| End Page: | 45529 |
| Language: | English |
| DOI: | 10.1074/jbc.M107615200 |
| PUBMED: | 11553638 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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