Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

Abstract

Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteinesulfinic acid and cysteine-sulfenic acid, catalyzing the hydration of nitriles to amides. Recently, we found that the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking advantage of the slow reactivity of tBuNC and the photoreactivity of nitrosylated NHase, we present the first structural evidence for the catalytic mechanism of NHase with time-resolved x-ray crystallography. By monitoring the reaction with attenuated total reflectanceFourier transform infrared spectroscopy, the product from the isonitrile carbon was identified as a CO molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and stopped by flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron center and then coordinated to the iron ion at 120 min. At 440 min, the electron density of tBuNC was significantly altered, and a new electron density was observed near the isonitrile carbon as well as the sulfenate oxygen of ␣Cys 114. These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by ␣Cys -SOH.Nitrile hydratases (NHases) 2 catalyze the hydration of nitriles to the corresponding amides and are used as catalysts in the production of acrylamide, making them one of the most important industrial enzymes (1, 2). NHases contain a nonheme Fe 3ϩ or non-corrin Co 3ϩ catalytic center. Iron-type NHases show unique photoreactivity; the enzyme is inactivated by nitrosylation in the dark and immediately reactivated by photo-induced denitrosylation (3-5). The protein structure is highly conserved among all known NHases (6 -9) as well as a related enzyme, thiocyanate hydrolase (10). The metal site is also conserved, with a distorted octahedral geometry. All ligand residues are involved in a strictly conserved motif of the ␣ subunit, Cys 1 -Xaa-Leu-Cys 2 -Ser-Cys 3 , where two amide nitrogens of Ser and Cys 3 and three Cys sulfurs are coordinated to the metal (6). Cys 2 and Cys 3 are post-translationally modified to cysteine-sulfinic acid and cysteine-sulfenic acid, respectively (7), which probably take deprotonated forms at the metal site (11). The sixth ligand site is occupied by a solvent molecule (8) or by a NO molecule in nitrosylated iron-type NHase (7).Several reaction mechanisms have been proposed based on the protein structures (1, 6). First, nitriles directly bind to the metal to facilitate the nucleophilic attack of a water molecule on the nitrile carbon. In the other mechanisms, a water molecule activated by the metal directly or indirectly attacks nitriles trapped near the metal. In all cases, the metal is suspected to function as a Lewis acid. By reconstituting iron-type NHase from recombinant unmodified subunits, we demonstrated that the post-translational ...

A QM/MM study of the initial steps of catalytic mechanism of nitrile hydratase

“…Gumataotao et al showed evidence of the intermediate species using single turnover stopped-flow spectroscopy [18]. Hashimoto et al analyzed the reaction mechanism of tert-butylisonitrile (tBuNC) conversion catalyzed by NHase using time-resolved X-ray crystallography and suggested that the substrate was coordinated to the iron ion and attacked by a water molecule activated by Cys-SO (inner-sphere mechanism) [19]. Martinez et al have shown that the X-ray crystal structures of Co-containing NHase with bronic acids (inhibitors) and suggested that the oxygen atom in the cysteine-sulfenic acid could act as a nucleophile and directly react with the substrate [20].…”

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

“…6) (6,16,(22)(23)(24). The rate constants provided herein suggest a fast second-order step that involves binding of substrate to the enzyme followed by rearrangement and then product release, which is the rate-limiting step.…”

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

“…Alternative mechanisms have also found support from time-resolved crystallographic and kinetic studies that involve an activated water molecule attacking the metal-bound nitrile (Fig. 1C) (7,8).…”