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Article: Aptamer-mediated inhibition of mycobacterium tuberculosis polyphosphate kinase 2

TitleAptamer-mediated inhibition of mycobacterium tuberculosis polyphosphate kinase 2
Authors
Issue Date2011
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry
Citation
Biochemistry, 2011, v. 50 n. 15, p. 3261-3271 How to Cite?
AbstractInorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzymes catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC 50 of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC 50 of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2. © 2011 American Chemical Society.
Persistent Identifierhttp://hdl.handle.net/10722/133577
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
Funding AgencyGrant Number
Hong Kong UGC GRFHKU 776507M
HKU 705007P
HKU200511159190
200411159146
Funding Information:

This work was supported by Hong Kong UGC GRF Grants HKU 776507M (to J.A.T.) and HKU 705007P (to R.M.W.) and by HKU Seed Funding Program for Basic Research Grants 200511159190 and 200411159146 (to J.A.T.).

References
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DC FieldValueLanguage
dc.contributor.authorShum, KTen_HK
dc.contributor.authorLui, ELHen_HK
dc.contributor.authorWong, SCKen_HK
dc.contributor.authorYeung, Pen_HK
dc.contributor.authorSam, Len_HK
dc.contributor.authorWang, Yen_HK
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorTanner, JAen_HK
dc.date.accessioned2011-05-24T02:11:11Z-
dc.date.available2011-05-24T02:11:11Z-
dc.date.issued2011en_HK
dc.identifier.citationBiochemistry, 2011, v. 50 n. 15, p. 3261-3271en_HK
dc.identifier.issn0006-2960en_HK
dc.identifier.urihttp://hdl.handle.net/10722/133577-
dc.description.abstractInorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzymes catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC 50 of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC 50 of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2. © 2011 American Chemical Society.en_HK
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_HK
dc.relation.ispartofBiochemistryen_HK
dc.subject.meshAptamers, Nucleotide - chemistry - genetics - metabolism - pharmacology-
dc.subject.meshEnzyme Inhibitors - chemistry - metabolism - pharmacology-
dc.subject.meshMycobacterium tuberculosis - enzymology-
dc.subject.meshPhosphotransferases (Phosphate Group Acceptor) - antagonists and inhibitors - chemistry - genetics - metabolism-
dc.subject.meshProtein Conformation-
dc.titleAptamer-mediated inhibition of mycobacterium tuberculosis polyphosphate kinase 2en_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0006-2960&volume=50&issue=15&spage=3261&epage=3271&date=2011&atitle=Aptamer-mediated+inhibition+of+Mycobacterium+tuberculosis+polyphosphate+kinase+2-
dc.identifier.emailWatt, RM:rmwatt@hku.hken_HK
dc.identifier.emailTanner, JA:jatanner@hku.hken_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.identifier.authorityTanner, JA=rp00495en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1021/bi2001455en_HK
dc.identifier.pmid21381755-
dc.identifier.scopuseid_2-s2.0-79953866877en_HK
dc.identifier.hkuros185113en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79953866877&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume50en_HK
dc.identifier.issue15en_HK
dc.identifier.spage3261en_HK
dc.identifier.epage3271en_HK
dc.identifier.isiWOS:000289319700022-
dc.publisher.placeUnited Statesen_HK
dc.relation.projectPolyphosphate metabolic enzymes as potential targets for anti-tuberculosis and general antibiotic drug development-
dc.relation.projectIdentification, validation and characterization of the eukaryotic enzymes and regulators of inorganic polyphosphate metabolism-
dc.identifier.scopusauthoridShum, KT=20436474600en_HK
dc.identifier.scopusauthoridLui, ELH=48662358900en_HK
dc.identifier.scopusauthoridWong, SCK=48663287100en_HK
dc.identifier.scopusauthoridYeung, P=35081534000en_HK
dc.identifier.scopusauthoridSam, L=6602908601en_HK
dc.identifier.scopusauthoridWang, Y=48662984600en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridTanner, JA=35513993000en_HK

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