Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330±20μM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.
- capping enzyme
- guanylate transferase
- mRNA capping
- RNA triphosphatase
Abbreviations used: AcMNPV, Autographa californica multinucleocapsid nucleopolyhedrovirus; BmLEF4, B. mori LEF4; BmNPV, Bombyx mori nucleopolyhedrovirus; DTT, dithiothreitol; GST, glutathione S-transferase; IPTG, isopropyl β-d-thiogalactoside; lef, LEF, gene and protein for late gene expression factor respectively; ORF, open reading frame; PBCV-1, Paramecium bursaria Chlorella virus 1; TCA, trichloracetic acid.
- The Biochemical Society, London ©2002