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 IC50 values of 30 and 41 nM, respectively. (C) 2010 Elsevier Inc. All rights reserved.