PDBsum entry 1wn7

Transferase

PDB id: 1wn7

Contents

Protein chain

729 a.a. *

Ligands

GOL

Metals

_NI

Waters ×277

* Residue conservation analysis

PDB id:

1wn7

Name:

Transferase

Title:

Crystal structure of archaeal family b DNA polymerase mutant

Structure:

Family b DNA polymerase. Chain: a. Engineered: yes

Source:

Thermococcus kodakarensis. Organism_taxid: 69014. Strain: kod1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.75Å R-factor: 0.226 R-free: 0.306

Authors:

T.Kuroita,H.Matsumura,N.Yokota,H.Hashimoto,T.Imanaka,T.Inoue,Y.Kai

Key ref:

T.Kuroita et al. (2005). Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases. J Mol Biol, 351, 291-298. PubMed id: 16019029 DOI: 10.1016/j.jmb.2005.06.015

Date:

28-Jul-04 Release date: 02-Aug-05

Protein chain

D0VWU9  (D0VWU9_THEKO) -  DNA polymerase from Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)

Seq: 774 a.a.

Struc: 729 a.a.

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

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

Added reference

DOI no: 10.1016/j.jmb.2005.06.015

J Mol Biol 351:291-298 (2005)

PubMed id: 16019029

Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases.

T.Kuroita, H.Matsumura, N.Yokota, M.Kitabayashi, H.Hashimoto, T.Inoue, T.Imanaka, Y.Kai.

ABSTRACT

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.

Selected figure(s)

Figure 1.
Figure 1. Overall structure of KOD DNA polymerase. The structure is composed of domains and subdomains, namely the N-terminal domain (N-ter, violet), exonuclease domain (Exo, gray) including the unique loop (blue), polymerase domain (Pol) including the Palm (brown) and Fingers (green) subdomains, and the Thumb domain (red), including the Thumb-1 and Thumb-2 subdomains and disordered regions (dotted lines). The orange region represents the E-cleft. The positions of two amino acids in the exonuclease domain are shown as colored balls (red, AA147; light blue, AA142).

Figure 2.
Figure 2. Amino acid sequence alignments of proposed structurally corresponding residues of KOD DNA polymerases (138-152, exonuclease domain; and 682-697, Thumb domain). Acidic, basic and hydrophobic residues are shown in red, blue and green, respectively. The boxed amino acid residues comprise the loop structure in the exonuclease domain. The underlined amino acid residues are the core sequence of the Exo I motif. The abbreviations used are as follows: KOD, Thermococcus kodakaraensis DNA polymerase; Tgo, Thermococcus gorgonarius DNA polymerase; Tli, Thermococcus litoralis DNA polymerase; 9°N-7, Thermococcus sp. 9°N-7 DNA polymerase; Tag, Thermococcus aggregans DNA polymerase; Pfu, Pyrococcus furiosus DNA polymerase; RB69, bacteriophage RB69 DNA polymerase; T7, bacteriophage T7 DNA polymerase.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 351, 291-298) copyright 2005.

Figures were selected by an automated process.