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dc.contributor.authorMattayaus Yentongchaien_US
dc.contributor.authorNiramon Thamwiriyasatien_US
dc.contributor.authorChompounoot Imtongen_US
dc.contributor.authorHui Chun Lien_US
dc.contributor.authorChanan Angsuthanasombaten_US
dc.contributor.otherTzu Chi Universityen_US
dc.contributor.otherMahidol Universityen_US
dc.contributor.otherBurapha Universityen_US
dc.contributor.otherPrince of Songkla Universityen_US
dc.contributor.otherBiophysics Institute for Research and Development (BIRD)en_US
dc.date.accessioned2020-11-18T07:59:33Z-
dc.date.available2020-11-18T07:59:33Z-
dc.date.issued2020-11-15en_US
dc.identifier.citationArchives of Biochemistry and Biophysics. Vol.694, (2020)en_US
dc.identifier.issn10960384en_US
dc.identifier.issn00039861en_US
dc.identifier.other2-s2.0-85092200778en_US
dc.identifier.urihttp://repository.li.mahidol.ac.th/dspace/handle/123456789/59854-
dc.description.abstract© 2020 Elsevier Inc. We previously demonstrated that the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys983 and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (KM and kcat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C14:0) and palmitoyl (C16:0) substrates of which catalytic efficiencies (kcat/KM) were roughly the same (~1.5 × 103 s−1mM−1). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys983, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser30 and His33 residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in kcat/KM of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser30-His33 nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.en_US
dc.rightsMahidol Universityen_US
dc.source.urihttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85092200778&origin=inwarden_US
dc.subjectBiochemistry, Genetics and Molecular Biologyen_US
dc.titlePreferential modification of CyaA-hemolysin by CyaC-acyltransferase through the catalytic Ser<sup>30</sup>-His<sup>33</sup> dyad in esterolysis of palmitoyl-donor substrate devoid of acyl carrier proteinsen_US
dc.typeArticleen_US
dc.rights.holderSCOPUSen_US
dc.identifier.doi10.1016/j.abb.2020.108615en_US
dc.identifier.urlhttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85092200778&origin=inwarden_US
Appears in Collections:Scopus 2020

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