Mutational analysis of a helicase motif-based RNA 5'-triphosphatase/NTPase from bamboo mosaic virus.
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.
Han
Yu-Tsung
YT
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung, Taiwan 40227, ROC.
Tsai
Chia-Sheng
CS
Chen
Ya-Chio
YC
Lin
Ming-Kuem
MK
Hsu
Yau-Heiu
YH
Meng
Menghsiao
M
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D027465:Chenopodium quinoa / Q000821:virology; D004252:DNA Mutational Analysis; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D017863:Potexvirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D031787:Sasa / Q000821:virology
D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases