PDBsum entry 2hr1

Transferase/DNA

PDB id: 2hr1

Contents

Protein chain

327 a.a. *

DNA/RNA

Ligands

SAH

Waters ×164

* Residue conservation analysis

PDB id:

2hr1

Name:

Transferase/DNA

Title:

Ternary structure of wt m.Hhai c5-cytosine DNA methyltransferase with unmodified DNA and adohcy

Structure:

5'-d( Gp Ap Tp Ap Gp Cp Gp Cp Tp Ap Tp C)-3'. Chain: c. Engineered: yes. 5'-d( Tp Gp Ap Tp Ap Gp Cp Gp Cp Tp Ap Tp C)-3'. Chain: d. Engineered: yes. Modification methylase hhai. Chain: a. Synonym: cytosine-specific methyltransferase hhai, m.Hhai.

Source:

Synthetic: yes. Haemophilus parahaemolyticus. Organism_taxid: 735. Gene: hhaim. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

1.96Å R-factor: 0.238 R-free: 0.269

Authors:

F.Shieh

Key ref:

F.K.Shieh et al. (2006). The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI. J Mol Biol, 362, 516-527. PubMed id: 16926025 DOI: 10.1016/j.jmb.2006.07.030

Date:

19-Jul-06 Release date: 19-Sep-06

Protein chain

P05102  (MTH1_HAEPH) -  Type II methyltransferase M.HhaI from Haemophilus parahaemolyticus

Seq: 327 a.a.

Struc: 327 a.a.

DNA/RNA chains

G-A-T-A-G-C-G-C-T-A-T-C 12 bases

T-G-A-T-A-G-C-G-C-T-A-T-C 13 bases

Enzyme reactions

• Enzyme class: E.C.2.1.1.37 - Dna (cytosine-5-)-methyltransferase.
• Reaction: a 2'-deoxycytidine in DNA + S-adenosyl-L-methionine = a 5-methyl- 2'-deoxycytidine in DNA + S-adenosyl-L-homocysteine + H⁺

2'-deoxycytidine in DNA + S-adenosyl-L-methionine = 5-methyl- 2'-deoxycytidine in DNA + S-adenosyl-L-homocysteine + H(+) (Bound ligand (Het Group name = SAH) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1016/j.jmb.2006.07.030

J Mol Biol 362:516-527 (2006)

PubMed id: 16926025

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI.

F.K.Shieh, B.Youngblood, N.O.Reich.

ABSTRACT

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.

Selected figure(s)

Figure 1.
Figure 1. (a) Stereo view showing superimposed WT M.HhaI (grey 3MHT.pdb) and R165A M.HhaI (cyan) complexed to AdoHcy (yellow in WT, orange in R165A), a DNA substrate (red in WT, violet in R165A) and Val121, Arg165, and Glu119 in green in the WT M.HhaI complex structure and in marine in the mutant complex structure. The flipped out cytosine (red in WT, violet in R165A) can be seen. The RMSD for both structures is 0.47 Å, based on comparing the protein backbones. (b) The previously characterized base flipping motif within the WT M.HhaI^26 showing contacts (dotted black lines) between the Arg165 and the flipped out cytosine base within the active site. DNA (magenta), and the flipped out cytosine base (salmon); Val121, Arg165, and Glu119 (green), and the protein backbone (gray) are shown. The distance unit is Å. (c) Stereo view shows superimposing the core active sites of the WT M.HhaI/DNA/AdoHcy (3MHT.pdb grey) and R165A M.HhaI/DNA/AdoHcy (cyan) ternary structures. The flipped out cytosine base, Cys81, Glu119, Val121, Arg163, Arg165, and Ala165 (thick lines), and the other residues and protein backbone (transparent sticks and transparent cartoon loops) are shown. (d) Stereo view showing the flipped out cytosine base with the 2F[o]–F[c] map (mesh in orange) contoured at 1.0 σ in the R165A M.HhaI/DNA/AdoHcy complex structure. (e) Stereo view showing omit electron density maps contoured at 1.0 σ, where the flipped out cytosine base and the 5′ phosphate were omitted in the structure factor calculation. The heteroatom colors are: red, oxygen; blue, nitrogen; hot pink, phosphorus; purple, water; and carbon atoms are shown in gray in all images. Figure 1. (a) Stereo view showing superimposed WT M.HhaI (grey 3MHT.pdb) and R165A M.HhaI (cyan) complexed to AdoHcy (yellow in WT, orange in R165A), a DNA substrate (red in WT, violet in R165A) and Val121, Arg165, and Glu119 in green in the WT M.HhaI complex structure and in marine in the mutant complex structure. The flipped out cytosine (red in WT, violet in R165A) can be seen. The RMSD for both structures is 0.47 Å, based on comparing the protein backbones. (b) The previously characterized base flipping motif within the WT M.HhaI[4]^26 showing contacts (dotted black lines) between the Arg165 and the flipped out cytosine base within the active site. DNA (magenta), and the flipped out cytosine base (salmon); Val121, Arg165, and Glu119 (green), and the protein backbone (gray) are shown. The distance unit is Å. (c) Stereo view shows superimposing the core active sites of the WT M.HhaI/DNA/AdoHcy (3MHT.pdb grey) and R165A M.HhaI/DNA/AdoHcy (cyan) ternary structures. The flipped out cytosine base, Cys81, Glu119, Val121, Arg163, Arg165, and Ala165 (thick lines), and the other residues and protein backbone (transparent sticks and transparent cartoon loops) are shown. (d) Stereo view showing the flipped out cytosine base with the 2F[o]–F[c] map (mesh in orange) contoured at 1.0 σ in the R165A M.HhaI/DNA/AdoHcy complex structure. (e) Stereo view showing omit electron density maps contoured at 1.0 σ, where the flipped out cytosine base and the 5′ phosphate were omitted in the structure factor calculation. The heteroatom colors are: red, oxygen; blue, nitrogen; hot pink, phosphorus; purple, water; and carbon atoms are shown in gray in all images.

Figure 4.
Figure 4. Stereo views of the WT M.HhaI and R165A M.HhaI bound to cognate DNA, and the WT M.HhaI bound to DNA with constrained sugar analog substitution.^25 (a)1 The active site of the WT M.HhaI is shown with Cys81, Glu119, Val121, Arg163, Arg165, and AdoHcy, which have contacts (dotted red lines) to the flipped out cytosine base (3MHT.pdb). (a)2 The flipped out cytosine is positioned in the active site pocket in the WT M.HhaI protein. (b)1 The active site of the R165A M.HhaI is shown with Cys81, Glu119, Asn120, Val121, Arg163, Glu164, and Ala165, which have contacts to the flipped out cytosine base (dotted red lines). (b)2 The flipped out cytosine base is positioned in the active site pocket in the R165A M.HhaI protein. (c) The flipped out South-constrained abasic carbocyclic sugar is rotated in the active site pocket in the WT M.HhaI protein (1SKM.pdb).The distance unit is Å and the heteroatom colors are: red, oxygen; blue, nitrogen; orange, sulfur; hot pink, phosphorus. Figure 4. Stereo views of the WT M.HhaI and R165A M.HhaI bound to cognate DNA, and the WT M.HhaI bound to DNA with constrained sugar analog substitution.[3]^25 (a)1 The active site of the WT M.HhaI is shown with Cys81, Glu119, Val121, Arg163, Arg165, and AdoHcy, which have contacts (dotted red lines) to the flipped out cytosine base (3MHT.pdb). (a)2 The flipped out cytosine is positioned in the active site pocket in the WT M.HhaI protein. (b)1 The active site of the R165A M.HhaI is shown with Cys81, Glu119, Asn120, Val121, Arg163, Glu164, and Ala165, which have contacts to the flipped out cytosine base (dotted red lines). (b)2 The flipped out cytosine base is positioned in the active site pocket in the R165A M.HhaI protein. (c) The flipped out South-constrained abasic carbocyclic sugar is rotated in the active site pocket in the WT M.HhaI protein (1SKM.pdb).The distance unit is Å and the heteroatom colors are: red, oxygen; blue, nitrogen; orange, sulfur; hot pink, phosphorus.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 362, 516-527) copyright 2006.

Figures were selected by an automated process.