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

Ka Yan Chung, Hongping Dong, Alexander Theodore Chao, Pei-Yong Shi, Julien Lescar, Siew Pheng Lim

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m7GpppNm) 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 (kcat) 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 41nM, respectively.

Original languageEnglish (US)
Pages (from-to)52-60
Number of pages9
JournalVirology
Volume402
Issue number1
DOIs
StatePublished - Jun 2010
Externally publishedYes

Fingerprint

Dengue Virus
Methylation
RNA
sinefungin
Flavivirus
Dengue
Viral RNA
Methyltransferases
Homocysteine
Methionine
Inhibitory Concentration 50
methylcobalamin-coenzyme M methyltransferase
Enzymes

Keywords

  • Dengue
  • Methyltransferase
  • Processivity
  • Steady state kinetic parameters

ASJC Scopus subject areas

  • Virology

Cite this

Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus. / Chung, Ka Yan; Dong, Hongping; Chao, Alexander Theodore; Shi, Pei-Yong; Lescar, Julien; Lim, Siew Pheng.

In: Virology, Vol. 402, No. 1, 06.2010, p. 52-60.

Research output: Contribution to journalArticle

Chung, Ka Yan ; Dong, Hongping ; Chao, Alexander Theodore ; Shi, Pei-Yong ; Lescar, Julien ; Lim, Siew Pheng. / Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus. In: Virology. 2010 ; Vol. 402, No. 1. pp. 52-60.
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N2 - Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m7GpppNm) 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 (kcat) 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 41nM, respectively.

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