Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand.
FEMS Microbiol Lett. 2010 Mar;304(2):183-90. doi: 10.1111/j.1574-6968.2010.01896.x. Epub 2010 Jan 8.
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.
百日咳博德特氏菌产生的腺苷酸环化酶-溶血素毒素（CyaA）需要 CyaC-酰基转移酶的脂肪酸酰基修饰才能成为活性毒素。先前，在大肠杆菌中表达的重组 CyaA 孔形成（CyaA-PF）片段在体内被共合成的 CyaC 棕榈酰化后显示出溶血活性。在这里，分别在大肠杆菌中作为包涵体表达的 21kDa CyaC 酶在 8M 脲中溶解，并成功重折叠成具有酶活性的单体。除了在体外激活 CyaA-PF 的能力外，CyaC 还对 p-硝基苯乙酸酯（pNPA）和 p-硝基苯棕榈酸酯（pNPP）表现出酯酶活性，与糜蛋白酶相比，对 pNPP 的水解具有优先性。基于同源性的 CyaC 结构表明，催化三联体包括 Ser（30）、His（33）和 Tyr（66）可能在底物催化中发挥作用。这些单个残基的丙氨酸取代导致三种突变酶（S30A、H33A 和 Y66A）对 pNPP 的特异性活性急剧下降，表明 CyaC-酰基转移酶与丝氨酸酯酶具有相似的水解机制，其中 Ser（30）是催化三联体的一部分。
