DNA (cytosine-5-)-methyltransferase (HhaI)

 

Methyl transferase (MTase) M. Hhal is an m5C-MTase that forms part of a type II restriction-modification system from Haemophilus haemolyticus. The enzyme recognises the specific tetranucleotide sequence, 5-GCGC-3. DNA methylation is found in diverse organisms ranging from bacteria to mammals and plants. Cytosine-5-methyltransferases (m5CMTases) are involved in a variety of biological processes in prokaryotes and eukaryotes by catalysing the transfer of a methyl group from S-adenosyl- L-methionine (AdoMet) to the C5 position of cytosine. MTases exist as a component of restriction and modification systems in bacteria.

 

Reference Protein and Structure

Sequence
P05102 UniProt (2.1.1.37) IPR001525 (Sequence Homologues) (PDB Homologues)
Biological species
Haemophilus parahaemolyticus (Bacteria) Uniprot
PDB
1mht - COVALENT TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE, DNA AND S-ADENOSYL-L-HOMOCYSTEINE (2.6 Å) PDBe PDBsum 1mht
Catalytic CATH Domains
3.40.50.150 CATHdb (see all for 1mht)
Click To Show Structure

Enzyme Reaction (EC:2.1.1.37)

S-adenosyl-L-methionine zwitterion
CHEBI:59789ChEBI
+
2'-deoxycytidine 5'-monophosphate(1-) residue
CHEBI:85452ChEBI
→
S-adenosyl-L-homocysteine zwitterion
CHEBI:57856ChEBI
+
5-methyl dCMP(1-) residue
CHEBI:85454ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: EcoRI methylase, DNA 5-cytosine methylase, DNA cytosine c(5) methylase, DNA cytosine methylase, DNA methylase, DNA methyltransferase, DNA transmethylase, DNA-cytosine 5-methylase, DNA-cytosine methyltransferase, HpaII methylase, HpaII' methylase, M.BsuRIa, M.BsuRIb, Typ II DNA methylase, Cytosine 5-methyltransferase, Cytosine DNA methylase, Cytosine DNA methyltransferase, Cytosine-specific DNA methyltransferase, Deoxyribonucleate methylase, Deoxyribonucleate methyltransferase, Deoxyribonucleic (cytosine-5-)-methyltransferase, Deoxyribonucleic acid (cytosine-5-)-methyltransferase, Deoxyribonucleic acid methylase, Deoxyribonucleic acid methyltransferase, Deoxyribonucleic acid modification methylase, Deoxyribonucleic methylase, Methylphosphotriester-DNA methyltransferase, Modification methylase, Restriction-modification system, Site-specific DNA-methyltransferase (cytosine-specific), Type II DNA methylase, DNA cytosine C(5) methylase,

Enzyme Mechanism

Introduction

The ring of cytosine is delivered to the active site by "flipping" the base out of the helix and replacing inter-helix hydrogen bond interactions with protein-helix interactions [PMID:8293469, DOI:10.1021/ja00079a047]. The nucleophile deprotonated Cys81 attacks the C6 position of the cytosine. The negative charge on C5 then demethylates S-adenosyl-L-methionine, subsequent deprotonation of the C positions reforms the double bond and releases the enzyme, methylated DNA and S-adenosyl-L-homocysteine.

Catalytic Residues Roles

UniProt PDB* (1mht)
Glu119 Glu119A(C) There is uncertainty in the literature as to the specific role of Glu119. In this annotation, the residue is shown to promote a resonance structure which enhances the electrophilicity at the C6 position of the cytosine ring, rather than acting a a general acid during the reaction [PMID:20471982]. attractive charge-charge interaction, hydrogen bond acceptor, electrostatic stabiliser, increase electrophilicity
Cys81 Cys81A(C) Deprotonated Cys81 performs a nucleophilic attack at the C6 of cytosine to initiate methylation of DNA. covalently attached, hydrogen bond acceptor, nucleofuge, nucleophile, polar interaction, proton donor, proton acceptor
Arg163, Arg165 Arg163A(C), Arg165A(C) Help stabilise the reactive intermediates and transition states formed during the course of the reaction. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser, increase electrophilicity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, proton relay, intermediate formation, overall reactant used, bimolecular nucleophilic addition, enzyme-substrate complex formation, rate-determining step, bimolecular nucleophilic substitution, overall product formed, bimolecular elimination, native state of enzyme regenerated, enzyme-substrate complex cleavage

References

  1. Klimasauskas S et al. (1994), Cell, 76, 357-369. Hhal methyltransferase flips its target base out of the DNA helix. DOI:10.1016/0092-8674(94)90342-5. PMID:8293469.
  2. Aranda J et al. (2016), ACS Catal, 6, 3262-3276. Unraveling the Reaction Mechanism of Enzymatic C5-Cytosine Methylation of DNA. A Combined Molecular Dynamics and QM/MM Study of Wild Type and Gln119 Variant. DOI:10.1021/acscatal.6b00394.
  3. Zangi R et al. (2010), J Mol Biol, 400, 632-644. Mechanism of DNA Methylation: The Double Role of DNA as a Substrate and as a Cofactor. DOI:10.1016/j.jmb.2010.05.021. PMID:20471982.
  4. Vilkaitis G et al. (2001), J Biol Chem, 276, 20924-20934. The Mechanism of DNA Cytosine-5 Methylation: KINETIC AND MUTATIONAL DISSECTION OF HhaI METHYLTRANSFERASE. DOI:10.1074/jbc.m101429200. PMID:11283006.
  5. Erlanson DA et al. (1993), J Am Chem Soc, 115, 12583-12584. DNA methylation through a locally unpaired intermediate. DOI:10.1021/ja00079a047.

Step 1. The cytosine ring is thought to exist in the N4 amine, O3 enol tautomer [PMID:20471982]. The catalytic Cys81 is deprotonated by the substrate-cofactor DNA phosphate group.

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Catalytic Residues Roles

Residue Roles
Arg163A(C) hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Glu119A(C) hydrogen bond acceptor, electrostatic stabiliser, attractive charge-charge interaction
Arg165A(C) hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Cys81A(C) proton donor

Chemical Components

proton transfer, proton relay, intermediate formation, overall reactant used

Step 2. The cysteine thiolate attacks at the C6 position of the cytosine ring. The electrophilicity of this carbon is increased through interactions with Arg163 and Arg165, as shown by computational calculations [PMID:20471982].

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Catalytic Residues Roles

Residue Roles
Cys81A(C) polar interaction
Arg163A(C) hydrogen bond donor, electrostatic stabiliser, increase electrophilicity, attractive charge-charge interaction
Glu119A(C) hydrogen bond acceptor, electrostatic stabiliser, increase electrophilicity, attractive charge-charge interaction
Arg165A(C) hydrogen bond donor, electrostatic stabiliser, increase electrophilicity, attractive charge-charge interaction
Cys81A(C) nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, enzyme-substrate complex formation, rate-determining step

Step 3. The carbanion residing on C5 attacks the methyl group of SAM. This results in methylation of the cytosine ring.

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Catalytic Residues Roles

Residue Roles
Cys81A(C) covalently attached
Arg163A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Glu119A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond acceptor
Arg165A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation

Step 4. Elimination of the Cys81 side chain involves interactions with a bridging water molecule which facilitates the elimination of the syn related proton, leaving a double bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys81A(C) hydrogen bond acceptor
Arg163A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Glu119A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond acceptor
Arg165A(C) attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Cys81A(C) proton acceptor, nucleofuge

Chemical Components

ingold: bimolecular elimination, proton transfer, overall product formed, native state of enzyme regenerated, enzyme-substrate complex cleavage, proton relay
Download: Image, Marvin File

Step 1. The cytosine ring is thought to exist in the N4 amine, O3 enol tautomer [PMID:20471982]. The catalytic Cys81 is deprotonated by the substrate-cofactor DNA phosphate group.

Step 2. The cysteine thiolate attacks at the C6 position of the cytosine ring. The electrophilicity of this carbon is increased through interactions with Arg163 and Arg165, as shown by computational calculations [PMID:20471982].

Step 3. The carbanion residing on C5 attacks the methyl group of SAM. This results in methylation of the cytosine ring.

Step 4. Elimination of the Cys81 side chain involves interactions with a bridging water molecule which facilitates the elimination of the syn related proton, leaving a double bond.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Anna Waters, Craig Porter, Charity Hornby