Human DNMT1 transition state structure

Abstract

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys 1226 to cytosine (Cyt) C6, methyl transfer from S-adenosyl-L-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys 1226 attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.H uman DNA methyltransferases (DNMTs) catalyze the formation of 5-methylcytosine (5mC) at CpG sites on DNA, a key epigenetic mark present in the human genome (1). DNA methylation is involved in transcriptional silencing, cellular differentiation, genomic imprinting, and X-chromosome inactivation. In addition, hypermethylation of CpG islands at gene promoter regions has been associated with carcinogenesis (2). Maintenance of DNA methylation patterns is conducted by human DNMT1, a multidomain protein of 1,616 amino acids. The C-terminal methyltransferase domain shows sequence similarities to the bacterial methyltransferases (3). Crystal structures of mouse and human DNMT1 complexed with different substrates have provided a structural basis for DNMT1-mediated maintenance DNA methylation (4, 5). Domain interactions and large conformational changes are responsible for properly positioning hemimethylated DNA within the active site and catalyze methyl transfer from S-adenosyl-L-methionine (SAM) to DNA. Site-directed mutations have offered insights into the structure-function relationship of DNMTs (6, 7), but their transition state (TS) structures have remained unknown. DNMT1 has been proposed to follow a catalytic mechanism shared by bacterial DNA-(cytosine C5)-methyltransferases (4, 8-10): nucleophilic attack of cytosine (Cyt) C6 by Cys 1226 of DNMT1, methyl transfer from SAM to Cyt C5, and β-elimination of H5 to produce 5mC in the final step (Fig. 1). Recent quantum mechanics (QM)/molecular mechanics (MM) and molecular dynamics (MD) simulations of the bacterial M.HhaI methyltransferase suggested that Cys 1226 attack is concerted with methyl transfer (11,12), and that β-elimination of H5 is the rate-limiting step (12). The combination of kinetic isotope effects (KIEs) and computational chemistry can test predicted reaction mechanisms and can provide a model of the T...

Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase

“…This primary methyl - 14 C KIE is consistent with values reported for NSD2, 14 DNMT1, 15 and glycine N -methyltransferase (GNMT). 21 The large magnitude of this KIE indicates that methyl transfer for hPNMT proceeds via a rate-limiting S N 2 mechanism.…”

“…13 Previously, our lab reported TS structures for the human protein lysine methyltransferase NSD2 14 and human DNA methyltransferase 1 (DNMT1). 15 In the current study, we extended these analyses to hPNMT by measuring intrinsic KIEs and generating a model for the TS structure of the methyl transfer reaction. Our model provides subangstrom detail of the hPNMT TS and supports methyl transfer as the rate-limiting step of an S N 2 process.…”

“…15 In this approach, SAM substrates selectively labeled with radioisotopes at either sensitive (“heavy”) or remote positions (“light”) are mixed and compete for the enzyme active site (Figure 1b and Table 1). Accordingly, V/K KIEs report on all steps of the reaction up to and including the first irreversible chemical step.…”

“…22 These isotope effects are in agreement with previous studies that support methyl transfer as the rate-limiting step of the PNMT reaction. 9,10 DFT calculations for NSD2 14 and DNMT1 15 suggest that the primary 36 S KIE on methyltransferase reactions is proportional to the loss of bond order between the carbon of the transferring methyl (C Me ) and the sulfur of SAM at the TS. The 36 S KIE for hPNMT (1.014 ± 0.005) is smaller than those measured for NSD2 (1.018 ± 0.008) 14 and DNMT1 (1.019 ± 0.006), 15 suggesting that hPNMT TS structure may retain somewhat greater C Me –S bond order.…”

“…9,10 DFT calculations for NSD2 14 and DNMT1 15 suggest that the primary 36 S KIE on methyltransferase reactions is proportional to the loss of bond order between the carbon of the transferring methyl (C Me ) and the sulfur of SAM at the TS. The 36 S KIE for hPNMT (1.014 ± 0.005) is smaller than those measured for NSD2 (1.018 ± 0.008) 14 and DNMT1 (1.019 ± 0.006), 15 suggesting that hPNMT TS structure may retain somewhat greater C Me –S bond order. This observation also suggests that the TS of hPNMT is early relative to those of NSD2 and DNMT1, though the 36 S KIEs for these three enzymes are similar within experimental error.…”