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Article: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

TitleStructural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
Authors
Issue Date2011
PublisherOxford 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?
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.
DescriptionEpub ahead of print; Open Access Journal
Persistent Identifierhttp://hdl.handle.net/10722/138889
ISSN
2013 Impact Factor: 8.808
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Ministry of Science and Technology of China2007CB914301
Research Grants Council of Hong Kong779109
University of Hong Kong
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.).

References

 

DC FieldValueLanguage
dc.contributor.authorYang, Wen_HK
dc.contributor.authorChen, WYen_HK
dc.contributor.authorWang, Hen_HK
dc.contributor.authorHo, JWSen_HK
dc.contributor.authorHuang, JDen_HK
dc.contributor.authorWoo, PCYen_HK
dc.contributor.authorLau, SKPen_HK
dc.contributor.authorYuen, KYen_HK
dc.contributor.authorZhang, Qen_HK
dc.contributor.authorZhou, Wen_HK
dc.contributor.authorBartlam, Men_HK
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorRao, Zen_HK
dc.date.accessioned2011-09-23T05:41:43Z-
dc.date.available2011-09-23T05:41:43Z-
dc.date.issued2011en_HK
dc.identifier.citationNucleic Acids Research, 2011, v. 39 n. 22, p. 9803-9819en_HK
dc.identifier.issn0305-1048en_HK
dc.identifier.urihttp://hdl.handle.net/10722/138889-
dc.descriptionEpub ahead of print; Open Access Journal-
dc.description.abstractAlkaline 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.en_HK
dc.languageengen_US
dc.publisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/en_HK
dc.relation.ispartofNucleic Acids Researchen_HK
dc.titleStructural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensisen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0305-1048&volume=&spage=&epage=&date=2011&atitle=Structural+and+functional+insight+into+the+mechanism+of+an+alkaline+exonuclease+from+Laribacter+hongkongensis-
dc.identifier.emailChen, WY: chenwy@hku.hken_HK
dc.identifier.emailWoo, PCY: pcywoo@hkucc.hku.hken_HK
dc.identifier.emailLau, SKP: skplau@hkucc.hku.hken_HK
dc.identifier.emailYuen, KY: kyyuen@hkucc.hku.hken_HK
dc.identifier.emailWatt, RM: rmwatt@hku.hken_HK
dc.identifier.authorityChen, WY=rp01487en_HK
dc.identifier.authorityWoo, PCY=rp00430en_HK
dc.identifier.authorityLau, SKP=rp00486en_HK
dc.identifier.authorityYuen, KY=rp00366en_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.description.naturepublished_or_final_versionen_US
dc.identifier.doi10.1093/nar/gkr660en_HK
dc.identifier.pmid21893587en_HK
dc.identifier.pmcidPMC3239189-
dc.identifier.scopuseid_2-s2.0-83755224379en_HK
dc.identifier.hkuros195552en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-83755224379&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume39en_HK
dc.identifier.issue22en_HK
dc.identifier.spage9803en_HK
dc.identifier.epage9819en_HK
dc.identifier.eissn1362-4962-
dc.identifier.isiWOS:000298186000035-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridYang, W=55305526800en_HK
dc.identifier.scopusauthoridChen, WY=37100973400en_HK
dc.identifier.scopusauthoridWang, H=36244769700en_HK
dc.identifier.scopusauthoridHo, JWS=7402650205en_HK
dc.identifier.scopusauthoridHuang, JD=54784706100en_HK
dc.identifier.scopusauthoridWoo, PCY=7201801340en_HK
dc.identifier.scopusauthoridLau, SKP=7401596211en_HK
dc.identifier.scopusauthoridYuen, KY=36078079100en_HK
dc.identifier.scopusauthoridZhang, Q=54785672300en_HK
dc.identifier.scopusauthoridZhou, W=7404515439en_HK
dc.identifier.scopusauthoridBartlam, M=6701775559en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridRao, Z=34668339000en_HK

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