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Mutational Probing of Catalytic and Rate-Limiting Steps in a DNA Cytosine-5 Methyltransferase
Summary: In high-resolution structural studies, Saulius Klimašauskas uses bacterial HhaI methyltransferase as a paradigm to probe the mechanisms of biological DNA methylation.
DNA methylation plays important roles in the regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigmatic bacterial methyltransferase 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 (kcat) of M.HhaI, and of other two cytosine-5 methyltransferases examined, is limited by a step subsequent to methyl transfer; however, no such step has yet 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; little variation is observed in the apparent methyl transfer rate kchem, as determined by rapid-quench experiments using immobilized fluorescently labeled DNA. However, Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher KDAdoMet and KMAdoMet), leading to faster turnover of the enzyme (10-fold higher kcat). 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.
Abstract from 2005 International Research Scholars Meeting
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