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

Abstract

Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using 18 O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles. Nitrile hydratases (NHases)2 catalyze the formation of amides from nitriles in aqueous, pH-neutral environments without any carboxylic acid byproducts (1, 2). These bacterial enzymes utilize trivalent iron or cobalt metal cations in this non-redox reaction, and uniquely, these metal ions are ligated directly to the protein backbone through amidate ligands and three cysteine sulfur ligands, of which two are post-translationally modified to the sulfinic and sulfenic acid ( Fig. 1A) (3). The mechanism of hydration is slowly emerging through multiple studies and appears to involve the sulfenic acid oxygen as the nucleophile. This sulfenate oxygen is proposed to initially attack the metal-bound nitrile carbon atom, creating a cyclic intermediate that is then resolved through attack at the sulfenic sulfur either directly by water or from the neighboring axial thiolato sulfur (Fig. 1B) (4 -6). 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).Early studies on sulfenato ligands in cobalt complexes demonstrated the nucleophilicity of the sulfenato oxygen (9, 10), and recently, this same characteristic was observed in NHases wherein the sulfenato oxygen reacted with electrophilic boronic acids (11). Quite tellingly, the activities of NHase, the related thiocyanate hydrolase, and NHase mimics were found to depend on the presence of these sulfenato ligands where oxidation to the sulfinate abrogated activity (12-14).To find direct evidence of either a protein-based or a solventbased nucleophile, we became interested in the observations made by Heinrich et al. (14) with an iron NHase mimic bearing two sulfenato ligands that is capable of hydrating nitriles with up to 50 turnovers. They discovered that labeled oxygen from solvent water was exchanged into one of these sulfenato groups only if the nitrile hydration reaction occurred. This provides a...

The 3rddegree of biomimetism: associating the cavity effect, ZnIIcoordination and internal base assistance for guest binding and activation

“…Nature provides nitrile hydratases, which are metallo-enzymes that combine the activation of the nitrilo substrate by coordination to Fe III or Co III to the attack of a coordinated sulfenate (derived from a cysteine residue) on either the water molecule (thus acting as an internal base) 37 , 38 or directly onto the substrate. 39 Although the biomimetic approach has led to the synthesis of a great variety of metal complexes, a relatively small number has been reported as active catalysts for the hydration of acetonitrile. 40 – 42 With mononuclear Co III complexes based on tetraaza ligands, simultaneous coordination of the nitrile and water molecules in the cis -position has been proposed to explain the efficiency of the catalyst.…”

Recent Advances and Promises in Nitrile Hydratase: From Mechanism to Industrial Applications

“…The LS state plays an important role in the binding and activation of nitrile substrates and to maintain the LS state, oxidized thiolate ligands are very crucial (Martinez et al, 2014). A recent report further proved that the posttranslational sulfonate acts as the nucleophile that initially attacks the nitrile, and found the source of the product carboxamide oxygen is from the protein (Figure 3D; Nelp et al, 2016). Further quantum mechanics investigation identified that the nitrile hydration reaction evolves toward the product in only three steps.…”

Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy

“… This unique chemistry was subsequently corroborated by time-resolved X-ray crystallography and DFT calculations. ,− Salient features of this mechanism include (i) direct coordination of the nitrile substrate to the active site metal center , and (ii) activation of the bound nitrile bond toward nucleophilic attack by the αCys–OH ligand, forming a cyclic intermediate ( I in Figure ). Subsequent 16/18 O mass spectrometric studies found that the product amide oxygen is protein-, rather than water-derived, providing further support for active participation by the sulfenic acid (αCys–OH) ligand. Once the cyclic intermediate is formed, it must react with H 2 O to generate the amide product, and two possible pathways have been proposed for this portion of the catalytic mechanism (Figure ).…”