Esterase activity of Bordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanism in vivo.
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.
Thamwiriyasati
Niramon
N
Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand.
Powthongchin
Busaba
B
Kittiworakarn
Jongrak
J
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001886:Bordetella pertussis / Q000201:enzymology*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D010168:Palmitates / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D037361:Adenylate Cyclase Toxin; D009596:Nitrophenols; D010168:Palmitates; D011994:Recombinant Proteins; C050015:4-nitrophenyl palmitate; C008642:4-nitrophenyl acetate; D000123:Acetyltransferases; D004950:Esterases