Substrate promiscuity in DNA methyltransferase M.PvuII. A mechanistic insight.
M.PvuII is a DNA methyltransferase from the bacterium Proteus vulgaris that catalyzes methylation of cytosine at the N4 position. This enzyme also displays promiscuous activity catalyzing methylation of adenine at the N6 position. In this work we use QM/MM methods to investigate the reaction mechanism of this promiscuous activity. We found that N6 methylation in M.PvuII takes place by means of a stepwise mechanism in which deprotonation of the exocyclic amino group is followed by the methyl transfer. Deprotonation involves two residues of the active site, Ser53 and Asp96, while methylation takes place directly from the AdoMet cofactor to the target nitrogen atom. The same reaction mechanism was described for cytosine methylation in the same enzyme, while the reversal timing, that is methylation followed by deprotonation, has been described in M.TaqI, an enzyme that catalyzes the N6-adenine DNA methylation from Thermus aquaticus. These mechanistic findings can be useful to understand the evolutionary paths followed by N-methyltransferases.
Departament de Química Física, Universitat de València, València, Spain.
Juan
J
Aranda
Maite
M
Roca
Iñaki
I
Tuñón
D000225:Adenine / Q000378:metabolism; D020134:Catalytic Domain; D003596:Cytosine / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000378:metabolism*; D008745:Methylation; D056004:Molecular Dynamics Simulation; D011514:Proteus vulgaris / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity
D003596:Cytosine; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases; D000225:Adenine