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

Abstract

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 K(AdoMet)D and K(AdoMet)M) 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.

Figure 1

Cofactor interactions in HhaI MTase. (A) The C_α trace of the HhaI–DNA–AdoHcy complex (PDB code 3mht); the closed catalytic loop is marked in blue and the open catalytic loop (from HhaI–AdoMet; 2hmy) is in light blue; DNA shown as dark red and flipped-out base is red; AdoHcy is orange. (B) Space filling models of the HhaI–DNA–AdoHcy complex (top) and the HhaI–AdoMet complex (bottom) revealing the position of the catalytic loop (blue) in the closed and open conformation, respectively, with respect to cofactor (orange) and Trp41 (gray). (C) A stick model of the AdoMet–Trp41 stacking interaction in the binary complex (2hmy); atom coloring: C is in gray, O in red, N in blue and S in yellow.

Figure 2

Kinetic analysis of Trp41 mutants. (A) Steady-state velocities for the mutants (circles, W41G; triangles, W41A; squares, W41F; diamonds, WT; stars, W41P) were determined at constant saturating poly(dG–dC) and varied [methyl-³H]AdoMet concentrations and the data were fitted to a Michaelis–Menten equation to reveal $K_{\text{M}}^{\text{AdoMet}}$ and k_cat (shown in Figure 3). (B) Single-turnover methylation reactions were performed by incubation of a biotin- and JOE-labeled 34mer DNA duplex with excess MTase and AdoMet in a rapid-quench-flow device. The DNA was immobilized and digested with R.Hin6I to release fluorescent fragments from unmethylated DNA. Fluorescence time courses (open circles, WT; filled circles, W41G) were fitted to a single exponential equation (solid lines) to reveal k_chem.

Figure 3

Cofactor binding and kinetic parameters of Trp41 mutants. (A) Comparison of binary ($K_{\text{D}}^{\text{AdoMet}}$) and ternary ($K_{\text{M}}^{\text{AdoMet}}$) MTase–cofactor interactions in the Trp41 mutants. (B) Comparison of a steady-state turnover rate k_cat and the rate of covalent methyl transfer k_chem in the Trp41 mutants.

Figure 4

Amino acid conservation of the Trp41 position (HhaI) in DNA C5-MTases is depicted, based on an alignment of 330 DNA C5-MTase sequences from the Pfam database (). (A) Representative sequences of the conserved motif II. Trp41 position (HhaI) is shown in boldface. Four degrees of conservation in descending order: black background with white text, dark gray background with white text, gray background with black text and white background with black text. (B) Distribution of amino acids found in the Trp41 position (HhaI) in DNA C5-MTases.

Figure 5

Breakdown of the ternary product complex. The minimal (A) and extended (B) kinetic mechanisms of the breakdown of the ternary product complex is shown. Acronyms describing molecular species are: E and E^L, open and closed (locked) forms of M.HhaI, respectively; D and mD, unmethylated and methylated DNA, respectively. Designation of events: dissociation of AdoHcy (i), opening of the catalytic loop (ii), departure (flip-back) of the target base from the active site (iii) and dissociation of the DNA (iv). (C) A log–log plot of $K_{\text{M}}^{\text{AdoMet}}$ versus k_cat with non-linear fits to mechanism A (broken line) and B (solid line).