Mutational analysis of a helicase motif-based RNA 5′-triphosphatase/NTPase from bamboo mosaic virus

Abstract

The helicase-like domain of BaMV replicase possesses NTPase and RNA 5'-triphosphatase activities. In this study, mutational effects of the helicase signature motifs and residue L543 on the two activities were investigated. Either activity was inactivated by K643A-S644A, D702A, D730A, R855A, or L543P mutations. On the other hand, Q826A, D858A and L543A had activities, in terms of k(cat)/K(m), reduced by 5- to 15-fold. AMPPNP, a nonhydrolyzable ATP analogue, competitively inhibited RNA 5'-triphosphatase activity. Analogies of mutational effects on the two activities and approximation of K(i(AMPPNP)) and K(m(ATP)) suggest that the catalytic sites of the activities are overlapped. Mutational effects on the viral accumulation in Chenopodium quinoa indicated that the activities manifested by the domain are required for BaMV survival. Results also suggest that Q826 in motif V plays an additional role in preventing tight binding to ATP, which would otherwise decrease further RNA 5'-triphosphatase, leading to demise of the virus in plant.

Function and Structural Organization of the Replication Protein of Bamboo mosaic virus

“…Adenylyl-imidodiphosphate (AMPPNP), a non-hydrolyzable ATP analog, is a competitive inhibitor of the RNA 5′-TPase activity. The inhibition constant K i (AMPPNP) was determined to be 93 μM, which is close to the K m value of ATP (150 μM) for the NTPase activity (Han et al, 2007). The closeness between the values of K i (AMPPNP) and K m (ATP) and the simultaneous inactivation of both activities by mutations at the featured motifs of helicases suggest that a common catalytic site is used for the hydrolysis of both NTP and RNA.…”

“…Both of these reactions required the presence of divalent Mg 2+ or Mn 2+ cations. Mutations at any of the signature motifs I, II, III, or VI of SF1 abrogate both types of activity (Han et al, 2007). Adenylyl-imidodiphosphate (AMPPNP), a non-hydrolyzable ATP analog, is a competitive inhibitor of the RNA 5′-TPase activity.…”

Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination

“…The function of the replicase linker region, where both mutations are located and their involvement in the virus recombination mechanism of the PVX replicase, as well as a possible involvement of host proteins remains elusive. Han et al (53) speculated that the potexvirus replicase linker might represent a flexible region that allows a dynamic interaction between MT and HEL during the replication process. We can only speculate that the pentapeptide insertion in the two replicase amino acid positions 430 and 434 interferes with this dynamic interaction necessary for a possible template switch of the molecule.…”

Identification and functional characterization of regions that can be crosslinked to RNA in the helicase-like domain of BaMV replicase

“…The BaMV helicase-like domain, generated as a fusion protein with a thioredoxin/hexahistidine/S-tag fused at the N-terminus, has been used to characterize associated enzymatic activities (Li et al, 2001;Han et al, 2007). Purification of the recombinant domain involved protein denaturation and refolding.…”

“…The BaMV helicaselike domain, also classified as SF1, exhibits ATPase and RTPase activities (Li et al, 2001). Inactivation of these two activities in response to mutations at the consensus residues within helicase motifs I, II, and VI and the competitive inhibition by an ATP analogue of RTPase activity suggest that the γ-phosphate of the two substrates is cleaved off by an identical set of catalytic residues (Han et al, 2007). However, it is reasonable to hypothesize that more residues are involved in RNA binding due to the much larger size of RNA than ATP.…”