Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study

The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S–O bond of αCys114–SO^– and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S–O bond via a hydrogen bond with the oxygen atom of αCys114–SO^–

Carbonyl Sulfide Hydrolase from <i>Thiobacillus thioparus</i> Strain THI115 Is One of the β‑Carbonic Anhydrase Family Enzymes

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. <i>Thiobacillus thioparus</i> strain THI115 can grow on thiocyanate (SCN^–) as its sole energy source. Previously, we showed that SCN^– is first converted to COS by thiocyanate hydrolase in <i>T. thioparus</i> strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H₂S and CO₂, the second step of SCN^– assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO₂ hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150–Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The <i>k</i>_cat/<i>K</i>_m value, 9.6 × 10⁵ s^–1 M^–1, is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, <i>in vitro</i> and <i>in vivo</i>. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS