Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus

Abstract

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.

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

“…The lack of inhibitory activity in these analogs was quite surprising, particularly because these compounds are very close derivatives of AdoHcy, which showed high potency in inhibition of the MTase activities in a number of studies [8,14,15,27]. These results prompted us to investigate whether AdoHcy is an effective inhibitor for flavivirus MTase.…”

“…Recently, flavivirus MTase became an attractive drug target due to its essential N-7 MTase function in viral replication [4–6,8–15,18,19,21,23,25,26,30]. AdoHcy, the by-product of the methyl transfer reaction, has been shown to inhibit both N-7 and 2’-O MTase activities for WNV, DENV2 and DENV3 [8,14,15,27]. The IC 50 values for inhibition of the WNV and DENV3 MTase activities by AdoHcy were estimated to be in low micromolar or even nanomolar range (from 0.34 µM to 3.19 µM) ( IC 50 : compound concentration required to inhibit enzyme activity by 50%).…”

“…Alternative higher throughput monitoring methods could possibly quantify non-specific binding of radiolabeled materials and/or signals arising from incorporation of radio-labeled materials to other positions of RNA. Previous studies employed the SPA-based scintillation assay in which [ 3 H]-AdoMet was used as a co-factor and activity was monitored by scintillation counting of the transfer of [ 3 H]-labeled methyl group to the viral RNA [8,14,15,27]. Non-specific binding of radio-labeled materials or incorporation of radio-labeled materials to positions other than N-7 and 2’-O of the RNA could affect the activity reported by this assay.…”

Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases

“…In the capping process, after the removal of the 59 cphosphate of RNA by RTPase, transfer of GMP occurs to the 59-diphosphate-ended RNA by GTase and then guanine (0.39 mM) are comparable with those of VV N 7 MTase (Chung et al, 2010). The lack of methylation in RNA in the absence of SAM or when using p127 with a mutant SAMbinding site (GLGVG) indicates that SAM plays a critical role in both N 7 and 29-O methylation.…”

Predicting Zika virus structural biology: Challenges and opportunities for intervention

“…The few reported viral MTase inhibitors exhibited low cell permeability, off-target activity, and poor drug-likeness. [41][42][43] Despite this, these compounds are likely potent inhibitors of ZIKV NS5 MTase and may serve as lead candidates in optimization studies.…”