Esterase activity ofBordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanismin vivo

Abstract

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

“…The weak reaction of grapevine leaf to the extract from E. coli expressing Xff S200A-LesA may result from residual lipase/esterase activity. Another serine esterase retained a few percent of the wild-type activity in an active site serine-to-alanine replacement mutant 40 .…”

Direct enzymatic acylation of cellulose pretreated in BMIMCl ionic liquid

“…Namely, lipases having a hydrophobic lid which covers the catalytic site of the enzyme are not able to function, in contrast to esterase and cutinase, which do not have this hydrophobic part and thus providing a more hydrophilic behaviour (Beer et al, 1996;Garcia et al, 2004;Guebitz and Cavacopaulo, 2008;Gundlach, 1973;Micaelo, 2005;Silva et al, 2005;Thamwiriyasati et al, 2010;Yamada et al, 1994). Besides, cellulose is by definition extremely hydrophilic because it is replete with hydroxyl groups.…”

Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen

“…Inclusions were collected by centrifugation and then solubilized in denaturing buffer (50 mM Na 2 HPO 4 , 300 mM NaCl, 8 M urea, pH 7.0) at 4 °C for 2 h. Solubilized nanobodies were purified using TALON™ Metal Affinity Resin (Clontech Laboratories, Mountain View, CA, USA) under denaturing conditions of 8 M urea. Refolding of the purified nanobodies was performed as described previously [ 26 ]. Specificity of refolded purified nanobodies to the CyaA-Hly toxin was verified by indirect ELISA.…”