Reconciling Structure and Function in HhaI DNA Cytosine-C-5 Methyltransferase

Abstract

Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme⅐DNA complex and the lack of catalytic competence of the enzyme⅐S-adenosyl-L-methionine (AdoMet) complex. The enzyme⅐AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme⅐AdoMet⅐DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme⅐AdoMet and ternary enzyme⅐S-adenosyl-Lhomocysteine⅐DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA. We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the 5-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi-and unmethylated substrates alter the fates and lifetimes of discrete enzyme⅐substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

“…Subsequent to its isolation from Hemophilus haemolyticus nearly a quarter century ago, HhaI DNA methyltransferase (M.HhaI) 1 has been subjected to incisive investigations yielding detailed insights into the reaction chemistry (3)(4)(5)(6)(7). M.HhaI is an S-adenosyl-Lmethionine (AdoMet) dependent DNA methyltransferase that catalyzes the covalent attachment of a methyl group at the C-5 position of the aromatic ring of the first cytosine in the specific sequence 5Ј-GCGC-3Ј.…”

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

“…Elucidating the kinetic mechanism of the reactions catalyzed by these enzymes still remains an important problem to investigate in the area of biological DNA methylation. Kinetic schemes have been proposed for HhaI (4 -7), MspI (8), human Dnmt1 (9), and murine Dnmt1 (10) [C 5 -cytosine]MTases, and for EcoRI (11,12), EcaI (13), TaqI (14), EcoRV (15), and Type III EcoP15I [N 6 -adenine]MTases (16). All of these enzymes exhibit a sequential bi-bi mechanism; however, they differ with respect to the order of substrate binding and ratelimiting step.…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…Proton elimination from the C5 atom of the methylated cytosine and β-elimination of the Cys residue result in a breakdown of the covalent DNA-protein complex and restore aromaticity of the modified cytosine base (Figure 2, a). The rate constant for methyl group transfer catalyzed by M.HhaI is about 0.14-0.26 s -1 [17,18]. The following release of the reaction products is a rate-limiting step of the M.HhaI catalytic cycle [17,18].…”

“…The rate constant for methyl group transfer catalyzed by M.HhaI is about 0.14-0.26 s -1 [17,18]. The following release of the reaction products is a rate-limiting step of the M.HhaI catalytic cycle [17,18].…”

“…The target cytosine is methylated according to S N 2 mechanism. The whole methylation process can be divided into 3 steps: a cytosine flipping out of the DNA double helix, a formation of a covalent enzyme-substrate intermediate and a methyl group transfer to the cytosine residue [14][15][16][17][18][19][20][21][22][23] (Figure 2). The target nucleotide flipping can occur spontaneously or with the help of an enzyme [20].…”