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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/59854
Title: Preferential 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 proteins
Authors: Mattayaus Yentongchai
Niramon Thamwiriyasati
Chompounoot Imtong
Hui Chun Li
Chanan Angsuthanasombat
Tzu Chi University
Mahidol University
Burapha University
Prince of Songkla University
Biophysics Institute for Research and Development (BIRD)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 15-Nov-2020
Citation: Archives of Biochemistry and Biophysics. Vol.694, (2020)
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.
URI: http://repository.li.mahidol.ac.th/dspace/handle/123456789/59854
metadata.dc.identifier.url: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85092200778&origin=inward
ISSN: 10960384
00039861
Appears in Collections:Scopus 2020

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