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.

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 Oct 01;:108615 Authors: Yentongchai M, Thamwiriyasati N, Imtong C, Li HC, Angsuthanasombat C 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 (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,...
Source: Archives of Biochemistry and Biophysics - Category: Biochemistry Authors: Tags: Arch Biochem Biophys Source Type: research