Summary information and primary citation
- PDB-id
- 3sz5; DSSR-derived features in text and JSON formats
- Class
- hydrolase-DNA
- Method
- X-ray (2.8 Å)
- Summary
- Crystal structure of lhk-exo in complex with 5-phosphorylated oligothymidine (dt)4
- Reference
- Yang W, Chen WY, Wang H, Ho JW, Huang JD, Woo PC, Lau SK, Yuen KY, Zhang Q, Zhou W, Bartlam M, Watt RM, Rao Z (2011): "Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis." Nucleic Acids Res., 39, 9803-9819. doi: 10.1093/nar/gkr660.
- Abstract
- Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.
