Mutational analysis of baculovirus capping enzyme Lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity.
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 K(m) for ATP (4 mm). Lef4 contains two conserved sequences, motif A ((8)IEKEISY(14)) and motif C ((180)LEYEF(184)), which define the fungal/viral/protozoal family of metal-dependent RNA triphosphatases. We find by mutational analysis that Glu(9), Glu(11), Glu(181), and Glu(183) 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.
Martins
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A
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
S
S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000201:enzymology*; D002413:Cations, Divalent; D004252:DNA Mutational Analysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D015335:Molecular Probes; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D002413:Cations, Divalent; D015335:Molecular Probes; D014764:Viral Proteins; C084398:late expression factor 4, baculovirus; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; C023090:RNA triphosphatase; D008274:Magnesium