Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-l-homocysteine (AdoHcy). The turnover rate (k(cat)) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate k(chem) as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher [Formula: see text] and [Formula: see text]) leading to a faster turnover of the enzyme (10-fold higher k(cat)). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme

Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-l-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events—the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems