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.
Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand.
Niramon
N
Thamwiriyasati
Busaba
B
Powthongchin
Jongrak
J
Kittiworakarn
Gerd
G
Katzenmeier
Chanan
C
Angsuthanasombat
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