Article: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
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- Publisher Website: 10.1093/nar/gkr660
- Scopus: eid_2-s2.0-83755224379
- PMID: 21893587
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| Title | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Authors | Yang, W2 Chen, WY3 Wang, H2 4 Ho, JWS5 Huang, JD1 Woo, PCY1 Lau, SKP1 Yuen, KY1 Zhang, Q2 Zhou, W2 Bartlam, M2 Watt, RM3 Rao, Z2 4 | ||||||||
| Issue Date | 2011 | ||||||||
| Publisher | Oxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/ | ||||||||
| Citation | Nucleic Acids Research, 2011, v. 39 n. 22, p. 9803-9819 [How to Cite?] DOI: http://dx.doi.org/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, 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. © The Author(s) 2011. Published by Oxford University Press. | ||||||||
| Description | Epub ahead of print; Open Access Journal | ||||||||
| ISSN | 0305-1048 2011 Impact Factor: 8.026 2011 SCImago Journal Rankings: 1.542 | ||||||||
| DOI | http://dx.doi.org/10.1093/nar/gkr660 | ||||||||
| ISI Accession Number ID | WOS:000298186000035
Funding Information: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.); General Research Fund (GRF) award from the Research Grants Council of Hong Kong (grant number 779109 to R.M.W.); Infection and Immunology Strategic Research Theme of the University of Hong Kong (to R.M.W.). Funding for open access charge: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.). | ||||||||
| PubMed Central ID | PMC3239189 | ||||||||
| References | References in Scopus |
| dc.contributor.author | Yang, W | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| dc.contributor.author | Chen, WY | ||||||||
| dc.contributor.author | Wang, H | ||||||||
| dc.contributor.author | Ho, JWS | ||||||||
| dc.contributor.author | Huang, JD | ||||||||
| dc.contributor.author | Woo, PCY | ||||||||
| dc.contributor.author | Lau, SKP | ||||||||
| dc.contributor.author | Yuen, KY | ||||||||
| dc.contributor.author | Zhang, Q | ||||||||
| dc.contributor.author | Zhou, W | ||||||||
| dc.contributor.author | Bartlam, M | ||||||||
| dc.contributor.author | Watt, RM | ||||||||
| dc.contributor.author | Rao, Z | ||||||||
| dc.date.accessioned | 2011-09-23T05:41:43Z | ||||||||
| dc.date.available | 2011-09-23T05:41:43Z | ||||||||
| dc.date.issued | 2011 | ||||||||
| dc.description.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, 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. © The Author(s) 2011. Published by Oxford University Press. | ||||||||
| dc.description.nature | published_or_final_version | ||||||||
| dc.description | Epub ahead of print; Open Access Journal | ||||||||
| dc.identifier.citation | Nucleic Acids Research, 2011, v. 39 n. 22, p. 9803-9819 [How to Cite?] DOI: http://dx.doi.org/10.1093/nar/gkr660 | ||||||||
| dc.identifier.doi | http://dx.doi.org/10.1093/nar/gkr660 | ||||||||
| dc.identifier.epage | 9819 | ||||||||
| dc.identifier.hkuros | 195552 | ||||||||
| dc.identifier.isi | WOS:000298186000035
Funding Information: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.); General Research Fund (GRF) award from the Research Grants Council of Hong Kong (grant number 779109 to R.M.W.); Infection and Immunology Strategic Research Theme of the University of Hong Kong (to R.M.W.). Funding for open access charge: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.). | ||||||||
| dc.identifier.issn | 0305-1048 2011 Impact Factor: 8.026 2011 SCImago Journal Rankings: 1.542 | ||||||||
| dc.identifier.issue | 22 | ||||||||
| dc.identifier.openurl | | ||||||||
| dc.identifier.pmcid | PMC3239189 | ||||||||
| dc.identifier.pmid | 21893587 | ||||||||
| dc.identifier.scopus | eid_2-s2.0-83755224379 | ||||||||
| dc.identifier.spage | 9803 | ||||||||
| dc.identifier.uri | http://hdl.handle.net/10722/138889 | ||||||||
| dc.identifier.volume | 39 | ||||||||
| dc.language | eng | ||||||||
| dc.publisher | Oxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/ | ||||||||
| dc.publisher.place | United Kingdom | ||||||||
| dc.relation.ispartof | Nucleic Acids Research | ||||||||
| dc.relation.references | References in Scopus | ||||||||
| dc.title | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis | ||||||||
| dc.type | Article |
Author Affiliations
- The University of Hong Kong Li Ka Shing Faculty of Medicine
- Nankai University
- Prince Philip Dental Hospital
- Tsinghua University
- Chinese University of Hong Kong