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
© 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 insigh...
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th-mahidol.598542020-11-18T14:59:33Z 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 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) Biochemistry, Genetics and Molecular Biology © 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. 2020-11-18T07:59:33Z 2020-11-18T07:59:33Z 2020-11-15 Article Archives of Biochemistry and Biophysics. Vol.694, (2020) 10.1016/j.abb.2020.108615 10960384 00039861 2-s2.0-85092200778 https://repository.li.mahidol.ac.th/handle/123456789/59854 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85092200778&origin=inward |
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Biochemistry, Genetics and Molecular Biology Mattayaus Yentongchai Niramon Thamwiriyasati Chompounoot Imtong Hui Chun Li Chanan Angsuthanasombat 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 |
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© 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. |
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Tzu Chi University |
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Tzu Chi University Mattayaus Yentongchai Niramon Thamwiriyasati Chompounoot Imtong Hui Chun Li Chanan Angsuthanasombat |
format |
Article |
author |
Mattayaus Yentongchai Niramon Thamwiriyasati Chompounoot Imtong Hui Chun Li Chanan Angsuthanasombat |
author_sort |
Mattayaus Yentongchai |
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 |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
publishDate |
2020 |
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https://repository.li.mahidol.ac.th/handle/123456789/59854 |
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1763492500020920320 |