Cargando…
Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
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 p...
Autores principales: | , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2011
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239189/ https://www.ncbi.nlm.nih.gov/pubmed/21893587 http://dx.doi.org/10.1093/nar/gkr660 |
_version_ | 1782219139395354624 |
---|---|
author | Yang, Wen Chen, Wen-yang Wang, Hui Ho, John W. S. Huang, Jian-Dong Woo, Patrick C. Y. Lau, Susanna K.P. Yuen, Kwok-Yung Zhang, Qionglin Zhou, Weihong Bartlam, Mark Watt, Rory M. Rao, Zihe |
author_facet | Yang, Wen Chen, Wen-yang Wang, Hui Ho, John W. S. Huang, Jian-Dong Woo, Patrick C. Y. Lau, Susanna K.P. Yuen, Kwok-Yung Zhang, Qionglin Zhou, Weihong Bartlam, Mark Watt, Rory M. Rao, Zihe |
author_sort | Yang, Wen |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3239189 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-32391892011-12-16 Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis Yang, Wen Chen, Wen-yang Wang, Hui Ho, John W. S. Huang, Jian-Dong Woo, Patrick C. Y. Lau, Susanna K.P. Yuen, Kwok-Yung Zhang, Qionglin Zhou, Weihong Bartlam, Mark Watt, Rory M. Rao, Zihe Nucleic Acids Res Structural Biology 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. Oxford University Press 2011-12 2011-09-05 /pmc/articles/PMC3239189/ /pubmed/21893587 http://dx.doi.org/10.1093/nar/gkr660 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Structural Biology Yang, Wen Chen, Wen-yang Wang, Hui Ho, John W. S. Huang, Jian-Dong Woo, Patrick C. Y. Lau, Susanna K.P. Yuen, Kwok-Yung Zhang, Qionglin Zhou, Weihong Bartlam, Mark Watt, Rory M. Rao, Zihe Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title_full | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title_fullStr | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title_full_unstemmed | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title_short | Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis |
title_sort | structural and functional insight into the mechanism of an alkaline exonuclease from laribacter hongkongensis |
topic | Structural Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239189/ https://www.ncbi.nlm.nih.gov/pubmed/21893587 http://dx.doi.org/10.1093/nar/gkr660 |
work_keys_str_mv | AT yangwen structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT chenwenyang structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT wanghui structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT hojohnws structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT huangjiandong structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT woopatrickcy structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT laususannakp structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT yuenkwokyung structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT zhangqionglin structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT zhouweihong structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT bartlammark structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT wattrorym structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis AT raozihe structuralandfunctionalinsightintothemechanismofanalkalineexonucleasefromlaribacterhongkongensis |