Preferential modification of CyaA-hemolysin by CyaC-acyltransferase through the catalytic Ser30-His33 dyad in esterolysis of palmitoyl-donor substrate devoid of acyl carrier proteins

Mattayaus Yentongchai; Niramon Thamwiriyasati; Chompounoot Imtong; Hui-Chun Li; Chanan Angsuthanasombat

doi:10.1016/j.abb.2020.108615

Preferential modification of CyaA-hemolysin by CyaC-acyltransferase through the catalytic Ser³⁰-His³³ dyad in esterolysis of palmitoyl-donor substrate devoid of acyl carrier proteins

Arch Biochem Biophys. 2020 Nov 15:694:108615. doi: 10.1016/j.abb.2020.108615. Epub 2020 Oct 2.

Authors

Mattayaus Yentongchai¹, Niramon Thamwiriyasati², Chompounoot Imtong³, Hui-Chun Li⁴, Chanan Angsuthanasombat⁵

Affiliations

¹ Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
² Molecular Medicine Research Group (MMRG), Division of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi, 20131, Thailand. Electronic address: niramon@go.buu.ac.th.
³ Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani, 94000, Thailand.
⁴ Department of Biochemistry, School of Medicine, Tzu Chi University, Hualian, 97004, Taiwan.
⁵ Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand; Department of Biochemistry, School of Medicine, Tzu Chi University, Hualian, 97004, Taiwan; Laboratory of Synthetic Biophysics and Chemical Biology, Biophysics Institute for Research and Development (BIRD), Chiang Mai, 50230, Thailand. Electronic address: chanan.ang@mahidol.ac.th.

PMID: 33011179
DOI: 10.1016/j.abb.2020.108615

Abstract

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 (K_M and k_cat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C_14:0) and palmitoyl (C_16:0) substrates of which catalytic efficiencies (k_cat/K_M) 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 k_cat/K_M 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.

Keywords: A nucleophile activation dyad; CyaC-acyltransferase; Esterolysis; Palmitoylation; Toxin activation; p-Nitrophenyl derivatives.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acyltransferases / chemistry*
Acyltransferases / genetics
Acyltransferases / metabolism
Adenylate Cyclase Toxin / chemistry*
Adenylate Cyclase Toxin / metabolism
Amino Acid Sequence
Bordetella pertussis / enzymology
Catalysis
Histidine / chemistry*
Kinetics
Lipoylation
Molecular Docking Simulation
Mutagenesis, Site-Directed
Mutation
Palmitates / chemistry*
Palmitates / metabolism
Protein Binding
Sequence Alignment
Serine / chemistry*
Substrate Specificity

Substances

Adenylate Cyclase Toxin
Palmitates
4-nitrophenyl palmitate
Serine
Histidine
Acyltransferases