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dc.contributor.authorAbdelhamid, Yassmin
dc.contributor.authorWang, Meng
dc.contributor.authorParkhill, Susannah L
dc.contributor.authorBrear, Paul
dc.contributor.authorChee, Xavier
dc.contributor.authorRahman, Md Taufiq
dc.contributor.authorWelch, Martin
dc.date.accessioned2021-12-15T12:14:10Z
dc.date.available2021-12-15T12:14:10Z
dc.date.issued2021
dc.date.submitted2021-10-07
dc.identifier.issn1664-302X
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/331506
dc.description.abstractPseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykF PA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the "ring loop" covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent "locked on" and "locked off" allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.
dc.languageen
dc.publisherFrontiers Media SA
dc.subjectMicrobiology
dc.subjectbacterial metabolism
dc.subjectEntner-Doudoroff pathway
dc.subjectglycolysis
dc.subjectPseudomonas aeruginosa
dc.subjectpyruvate kinase
dc.subjectpykF
dc.subjectx-ray crystallography
dc.titleStructure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa.
dc.typeArticle
dc.date.updated2021-12-15T12:14:09Z
prism.publicationNameFront Microbiol
prism.volume12
dc.identifier.doi10.17863/CAM.78960
dcterms.dateAccepted2021-10-26
rioxxterms.versionofrecord10.3389/fmicb.2021.790742
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidBrear, Paul [0000-0002-4045-0474]
dc.contributor.orcidRahman, Md Taufiq [0000-0001-7247-2013]
dc.contributor.orcidWelch, Martin [0000-0003-3646-1733]
dc.identifier.eissn1664-302X
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/M019411/1)
pubs.funder-project-idCystic Fibrosis Trust (SRC-017)
cam.issuedOnline2021-11-16


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