As the best-known feature of enzyme-catalyzed ester hydrolysis (Hardman et al., 1971) chymotrypsin was used as a control in the reaction. Table 2 shows that specific activities of the purified CyaC enzyme in catalyzing pNPA and pNPP are ∼49 U mg−1 and ∼289 U mg−1, respectively, indicating that CyaC exerted a much higher esterase activity toward a palmitoyl group, which has been shown

to be a preferred physiological substrate (Havlicek et al., 2001). Conversely, pNPA was preferred over pNPP for the chymotrypsin activity under the conditions used. We noted that both soluble and refolded CyaC showed relatively the same specific activity in catalyzing pNPA that was consistent with the CyaA-PF hemolytic activities HCS assay upon in vitro activation by either form of CyaC. Despite the fact that CyaC-acyltransferase and chymotrypsin exhibit different substrate preferences, their reactions toward these analogs may share a common feature regarding the hydrolysis of oxygen–ester bond. Therefore, structural insights into the mechanistic basis for the esterolytic reaction Smad inhibitor of CyaC in comparison with this serine esterase are of great interest. As the crystal structure of CyaC-acyltransferase has not been yet resolved, a plausible 3D structure of this enzyme was built instead by modeling

based on the known DABA structure, which is the best-fit template available so far in the acetyltransferase group. As shown in Fig. PIK3C2G 3, although pairwise alignment between DABA and CyaC displays only ∼30% sequence similarity, multiple alignments show relatively high similarity (∼50%) among all the nine related RTX-acyltransferases with the same template, implying a common 3D-folded structure for these

enzymes. Validating the model, its stereochemical quality showed an overall G-factor value of −0.15, which is in the range of good quality (the best model displaying a value close to 0) (Laskowski et al., 1996). The Ramachandran plot of the CyaC model revealed that over 90% of nonglycine and nonproline residues possess φ/ψ backbone-dihedral angles in energetically favorable and allowed regions. This indicates that the modeled structure has most of the sterically favorable main-chain conformations. As also assessed by CD spectroscopy, secondary structural contents of purified CyaC were found to be 25% helix and 27%β-strand, comparable to those estimated from the derived model (26% helix and 22%β-strand), supporting the validity of this model. As shown in Fig. 4a, the CyaC structure (Leu26-Ala185) comprises of a single domain with a β-sheet core of six strands (βA, βB, βC, βD, βE and βF) connected by five α-helices (αA, αB, αC, αD and αE) to form a two-layer α/β sandwich, which is a typical fold of α/β hydrolase family (Holmquist, 2000). Using molecular surface analysis, a hydrophobic groove was clearly visible in the CyaC structure (Fig. 4b).