PDBsum entry: 2i9k

Transferase/DNA

PDB id: 2i9k

Contents

Protein chain

327 a.a. *

DNA/RNA

Ligands

SAH

* Residue conservation analysis

PDB id:

2i9k

Name:

Transferase/DNA

Title:

Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase m.Hhai

Structure:

5'-d( T Gp Ap Tp Ap Gp Cp Gp Cp Tp Ap Tp C)-3'. Chain: c, d. Engineered: yes. Modification methylase hhai. Chain: a. Synonym: cytosine-specific methyltransferase hhai, m.Hhai. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Haemophilus haemolyticus. Organism_taxid: 726. Gene: hhaim. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.65Å R-factor: 0.238 R-free: 0.262

Authors:

B.Youngblood,F.K.Shieh,S.De Los Rios,J.J.Perona,N.O.Reich

Key ref:

B.Youngblood et al. (2006). Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI. J Mol Biol, 362, 334-346. PubMed id: 16919299 DOI: 10.1016/j.jmb.2006.07.031

Date:

05-Sep-06 Release date: 10-Oct-06

Protein chain

P05102  (MTH1_HAEPH) -  Modification methylase HhaI

Seq: 327 a.a.

Struc: 327 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.1.1.37 - Dna (cytosine-5-)-methyltransferase.
• Reaction: S-adenosyl-L-methionine + DNA = S-adenosyl-L-homocysteine + DNA containing 5-methylcytosine

S-adenosyl-L-methionine + DNA = S-adenosyl-L-homocysteine (Bound ligand (Het Group name = SAH) corresponds exactly) + DNA containing 5-methylcytosine

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

 Gene Ontology (GO) functional annotation 

• Biological process: DNA restriction-modification system (2 terms)
• Biochemical function: transferase activity (4 terms)

reference

DOI no: 10.1016/j.jmb.2006.07.031

J Mol Biol 362:334-346 (2006)

PubMed id: 16919299

Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI.

B.Youngblood, F.K.Shieh, S.De Los Rios, J.J.Perona, N.O.Reich.

ABSTRACT

Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.

Selected figure(s)

Figure 5.
Figure 5. Stereoview of M.HhaI cavities and residues 118–132 (orange) and residues 80–99 (green). (a) Stereoview of the difference map between F124A and WT M.HhaI showing a cavity in the position of residue 124, represented by the blue mesh. Ala124, His127, Thr132 (purple), Phe84 (pink), and the flipped out base (cyan). (b) Proximity of F124A M.HhaI residues and structural accommodations around the cavity (red). (c) Location of all cavities in the WT M.HhaI identified by VOIDOO using a 1.2 Å probe radius. The edge to edge distance between Phe124 and Phe84 is 3.5 Å. The flipped out base is cyan. The M.HhaI ternary structure (PDB ID 3MHT).

Figure 6.
Figure 6. Comparison of DNA cytosine C5 methyltransferase structures. Residue 124 for M.HhaI and the corresponding residue 114 for M.HaeIII are colored purple. Residue 84 for M.HhaI and the corresponding residue 74 for M.HaeIII are colored pink. (a) Alignment of M.HhaI, M.HaeIII, DNMT2, and the C termini of DNMT1. (b) Crystal structures of M.HhaI (PDB ID 3MHT), residues 75–145, and M.HaeIII (PDB ID 1DCT), residues 65–135.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 362, 334-346) copyright 2006.

Figures were selected by an automated process.