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Article: The functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase

TitleThe functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase
Authors
Issue Date2009
Citation
Journal Of Bacteriology, 2009, v. 191 n. 15, p. 4722-4731 How to Cite?
AbstractEAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α) 8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K m) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg 2+ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Persistent Identifierhttp://hdl.handle.net/10722/171778
ISSN
2015 Impact Factor: 3.198
2015 SCImago Journal Rankings: 2.216
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorRao, Fen_US
dc.contributor.authorQi, Yen_US
dc.contributor.authorChong, HSen_US
dc.contributor.authorKotaka, Men_US
dc.contributor.authorLi, Ben_US
dc.contributor.authorLi, Jen_US
dc.contributor.authorLescar, Jen_US
dc.contributor.authorTang, Ken_US
dc.contributor.authorLiang, ZXen_US
dc.date.accessioned2012-10-30T06:17:00Z-
dc.date.available2012-10-30T06:17:00Z-
dc.date.issued2009en_US
dc.identifier.citationJournal Of Bacteriology, 2009, v. 191 n. 15, p. 4722-4731en_US
dc.identifier.issn0021-9193en_US
dc.identifier.urihttp://hdl.handle.net/10722/171778-
dc.description.abstractEAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α) 8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K m) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg 2+ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution. Copyright © 2009, American Society for Microbiology. All Rights Reserved.en_US
dc.languageengen_US
dc.relation.ispartofJournal of Bacteriologyen_US
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshBacterial Proteins - Chemistry - Genetics - Metabolismen_US
dc.subject.meshBinding Sites - Geneticsen_US
dc.subject.meshComputational Biologyen_US
dc.subject.meshComputer Simulationen_US
dc.subject.meshCyclic Gmp - Analogs & Derivatives - Metabolismen_US
dc.subject.meshGenome, Bacterial - Genetics - Physiologyen_US
dc.subject.meshKineticsen_US
dc.subject.meshMagnesium - Metabolismen_US
dc.subject.meshMass Spectrometryen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshMutationen_US
dc.subject.meshPhosphoric Diester Hydrolases - Chemistry - Genetics - Metabolismen_US
dc.subject.meshProtein Binding - Geneticsen_US
dc.subject.meshProtein Structure, Secondaryen_US
dc.subject.meshProtein Structure, Tertiaryen_US
dc.subject.meshPseudomonas Aeruginosa - Enzymology - Geneticsen_US
dc.titleThe functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesteraseen_US
dc.typeArticleen_US
dc.identifier.emailKotaka, M:masayo@hku.hken_US
dc.identifier.authorityKotaka, M=rp00293en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1128/JB.00327-09en_US
dc.identifier.pmid19376848-
dc.identifier.scopuseid_2-s2.0-67749113278en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-67749113278&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume191en_US
dc.identifier.issue15en_US
dc.identifier.spage4722en_US
dc.identifier.epage4731en_US
dc.identifier.isiWOS:000267937000005-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridRao, F=24465697600en_US
dc.identifier.scopusauthoridQi, Y=24465712900en_US
dc.identifier.scopusauthoridChong, HS=35087971200en_US
dc.identifier.scopusauthoridKotaka, M=6604073578en_US
dc.identifier.scopusauthoridLi, B=7410083528en_US
dc.identifier.scopusauthoridLi, J=35261852300en_US
dc.identifier.scopusauthoridLescar, J=6603844493en_US
dc.identifier.scopusauthoridTang, K=35088979400en_US
dc.identifier.scopusauthoridLiang, ZX=23668102800en_US

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