Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2'-O methyltransferase activity in dengue virus.
Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m(7)GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2'-O methylations. Sequential N-7 to 2'-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme-RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2'-O methylation as it turnovers RNA faster (k(cat)) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2'-O MTase activity, exhibiting IC(50) values of 30 and 41nM, respectively.
Chung
Ka Yan
KY
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, 138670, Singapore.
Dong
Hongping
H
Chao
Alexander Theodore
AT
Shi
Pei-Yong
PY
Lescar
Julien
J
Lim
Siew Pheng
SP
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D003716:Dengue Virus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014764:Viral Proteins; D012313:RNA; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; C055099:mRNA (nucleoside-O(2'))-methyltransferase; D000241:Adenosine; C006235:sinefungin