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Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus

Sehrawat, Seema and Srinivasan, Narayanaswamy and Gopinathan, Karumathil P (2002) Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus. In: Biochemical Journal, 368 . pp. 159-169.

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Abstract

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 $K_m$ of $330 \pm 20\hspace{2mm}\mu M$. 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.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to The Biochemical Society.
Keywords: ATPase;Capping enzyme;Guanylate transferase;mRNA capping;RNA triphosphatase
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 23 Oct 2007
Last Modified: 19 Sep 2010 04:40
URI: http://eprints.iisc.ernet.in/id/eprint/12308

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