Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5.
Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.
Christian
Thomas
T
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Lahoud
Georges
G
Liu
Cuiping
C
Hoffmann
Katherine
K
Perona
John J
JJ
Hou
Ya-Ming
YM
D002384:Catalysis; D020134:Catalytic Domain; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000378:metabolism*
D012436:S-Adenosylmethionine; D012343:RNA, Transfer; D012359:tRNA Methyltransferases