N-terminal nucleophile (Ntn-) hydrolases are a superfamily of enzymes specialized in the cleavage of amide bonds 
. Ntn-hydrolases become catalytically active after autocatalytic cleavage of an N-terminal peptide, which creates a novel N-terminal residue – usually a Ser, Thr, or Cys – that is responsible for amide bond cleavage 
. As with classical amidases, Ntn-hydrolases are thought to catalyze the cleavage of their substrates by means of two consecutive reactions: (i) breakage of the amide bond with formation of an acylenzyme adduct; (ii) acylenzyme hydrolysis with regeneration of a catalytically competent enzyme.
Despite their common structural features, members of the Ntn-hydrolase family have evolved beyond any recognizable sequence homology 
. They include structurally and functionally different enzymes, such as penicillin V acylase (PVA), used to produce semi-synthetic penicillins 
, and conjugated bile acid hydrolase (CBAH), which is responsible for the hydrolysis of bile salts and controls the balance of cholesterol into the enterohepatic circulation 
. Ntn-hydrolase enzymes are also emerging as important targets for therapy. Notable examples include the subunit 20 S of the proteasome 
, which is inhibited by the anti-cancer drug bortezomib 
, N-acylethanolamine-hydrolyzing acid amidase (NAAA) 
, a potential target for anti-inflammatory and analgesic drugs 
, and acid ceramidase (AC) 
, a potential target for cancer chemosensitizing agents 
The crystal structure of CBAH from Clostridium perfringens
was resolved 
and recently used to build homology models of NAAA 
and AC 
. These models, which were validated by mutagenesis experiments 
, suggest that CBAH, NAAA, and AC share a relatively well-conserved active site. In addition to the N-terminal catalytic cysteine (Cys2, according to the CBAH sequence), other conserved residues appear to be essential for the amidase activity of these enzymes 
. These are Arg18 and Asp21, which undertake hydrogen bonds (H-bonds) with the sulfhydryl and alpha-amino groups of Cys2; Asn82 and Asn175, which form the putative oxyanion hole. Furthermore, kinetic experiments have shown that the hydrolytic activities of CBAH, NAAA, and AC display a bell-shaped pH dependency – with an optimal activity ranging from 4.5 for NAAA 
and AC 
, to nearly 6 for CBAH 
. These findings support the hypothesis that Ntn cysteine-hydrolases might have evolved to cleave their substrates using the same catalytic strategy.
A general mechanism for Ntn-hydrolases has been proposed by Oinonen and Rouvinen on the basis of structural data 
. According to their hypothesis, the N-terminal catalytic residue of these enzymes lives in the neutral form. The reaction starts with a proton transfer between the nucleophile (OH or SH) of the catalytic residue and the alpha-amino group of the same amino acid (). Once deprotonated, the nucleophile attacks the carbonyl carbon of the substrate, leading to the formation of the tetrahedral intermediate (TI). The reaction becomes complete when the alpha-amino group of the catalytic residue donates a proton to the nitrogen of the scissile amide bond. In this way, an acylenzyme adduct is formed and the amino leaving group of the substrate is released (). This mechanism is consistent with current knowledge of serine 
and threonine Ntn-hydrolases 
, but is in contrast with experimental evidence on cysteine Ntn-hydrolases. For instance, since the optimal activity of many cysteine Ntn-hydrolases occurs at acidic pH 
, it is unlikely that the alpha-amino group of the N-terminal Cys would act as a general base during the catalysis. In this respect, a recent study 
has suggested that the key cysteine of an isopenicillin N-converting Ntn-hydrolase participates in the catalysis in its zwitterionic form, with the N-terminus positively charged and an anionic thiolate group.
Catalytic mechanism of Ntn-hydrolases.
Understanding the catalytic mechanism of cysteine Ntn-hydrolases would be important for three main reasons: (i) to define, at an atomic level, the mechanism of this reaction; (ii) to identify active-site residues important for catalysis; (iii) to provide insights for the rational design of pharmacologically useful inhibitors 
. In the present study, to elucidate the catalytic mechanism of this class of enzymes, we investigated the first step of taurodeoxycholate (TAU) hydrolysis by CBAH () using a hybrid quantum mechanics/molecular mechanics (QM/MM) technique 
, a well-established approach in computational enzymology 
. Specifically, we calculated the free energy for TAU hydrolysis by using a conceptually innovative strategy based on enhanced sampling techniques, such as steered-molecular dynamics (steered-MD) and umbrella sampling (US) simulations, together with path collective variables (PCVs) 
. This computational approach has been shown to be effective in studying reaction mechanisms and to explicitly account for anharmonicity and entropic effects 
. Our analyses suggest that (i) the catalytic N-terminal cysteine participates in catalysis via its zwitterionic form; and (ii) the lowest free energy reaction pathway shows the formation of a zwitterionic tetrahedral adduct, in analogy with other cysteine hydrolases. Our calculations also reveal that the tetrahedral adduct is characterized by a cyclic “chair-like” structure, which might represent a significant signature of this class of enzyme.