Nitrile hydratases (NHases; ) are mononuclear

iron Iron () is a chemical element with Symbol (chemistry), symbol Fe (from la, Wikt:ferrum, ferrum) and atomic number 26. It is a metal that belongs to the first transition series and group 8 element, group 8 of the periodic table. It is, Abundanc ...

or non-corrinoid

cobalt Cobalt is a chemical element with the symbol Co and atomic number 27. As with nickel, cobalt is found in the Earth's crust only in a chemically combined form, save for small deposits found in alloys of natural meteoric iron. The free element, p ...

enzymes that catalyse the hydration of diverse nitriles to their corresponding amides

R-C≡N + H₂O → R-C(O)NH₂

Metal cofactor

In biochemistry,

is in general found in a

corrin Corrin is a heterocyclic compound. It is the parent macrocycle related to the substituted derivative that is found in vitamin B12. Its name reflects that it is the "core" of vitamin B12 (cobalamins).Nelson, D. L.; Cox, M. M. "Lehninger, Princi ...

ring, such as in vitamin B₁₂. Nitrile hydratase is one of the rare enzyme types that use cobalt in a non-corrinoid manner. The mechanism by which the cobalt is transported to NHase without causing toxicity is unclear, although a cobalt

permease The permeases are membrane transport proteins, a class of multipass transmembrane proteins that allow the diffusion of a specific molecule in or out of the cell in the direction of a concentration gradient, a form of facilitated diffusion. The per ...

has been identified, which transports cobalt across the cell membrane. The identity of the metal in the active site of a nitrile hydratase can be predicted by analysis of the sequence data of the alpha subunit in the region where the metal is bound. The presence of the amino acid sequence VCTLC indicates a Co-centred NHase and the presence of VCSLC indicates Fe-centred NHase.

Metabolic pathway

Nitrile hydratase and amidase are two hydrating and hydrolytic enzymes responsible for the sequential metabolism of

nitriles In organic chemistry, a nitrile is any organic compound that has a functional group. The prefix '' cyano-'' is used interchangeably with the term ''nitrile'' in industrial literature. Nitriles are found in many useful compounds, including met ...

in bacteria that are capable of utilising nitriles as their sole source of nitrogen and carbon, and in concert act as an alternative to nitrilase activity, which performs nitrile hydrolysis without formation of an intermediate primary amide. A sequence in genome of the choanoflagellate ''Monosiga brevicollis'' was suggested to encode for a nitrile hydratase. The ''M. brevicollis'' gene consisted of both the alpha and beta subunits fused into a single gene. Similar nitrile hydratase genes consisting of a fusion of the beta and alpha subunits have since been identified in several eukaryotic supergroups, suggesting that such nitrile hydratases were present in the last common ancestor of all eukaryotes.

Industrial applications

NHases have been efficiently used for the industrial production of

acrylamide Acrylamide (or acrylic amide) is an organic compound with the chemical formula CH2=CHC(O)NH2. It is a white odorless solid, soluble in water and several organic solvents. From the chemistry perspective, acrylamide is a vinyl-substituted primary ...

from

acrylonitrile Acrylonitrile is an organic compound with the formula and the structure . It is a colorless, volatile liquid although commercial samples can be yellow due to impurities. It has a pungent odor of garlic or onions. In terms of its molecula ...

on a scale of 600 000 tons per annum, and for removal of nitriles from wastewater. Photosensitive NHases intrinsically possess nitric oxide (NO) bound to the iron centre, and its photodissociation activates the enzyme.

Nicotinamide Niacinamide or Nicotinamide (NAM) is a form of vitamin B3 found in food and used as a dietary supplement and medication. As a supplement, it is used by mouth to prevent and treat pellagra (niacin deficiency). While nicotinic acid (niacin) may ...

is produced industrially by the hydrolysis of

3-cyanopyridine Nicotinonitrile or 3-cyanopyridine is an organic compound with the formula NCC5H4N. The molecule consists of a pyridine ring with a nitrile group attached to the 3-position. A colorless solid, it is produced by ammoxidation of 3-methylpyridine: ...

catalysed by the nitrile hydratase from ''

Rhodococcus rhodochrous ''Rhodococcus rhodochrous'' is a bacterium used as a soil inoculant in agriculture and horticulture. It is gram positive, in the shape of rods/ cocci, oxidase negative, and catalase positive. It is industrially produced to catalyse acryloni ...

'' J1, producing 3500 tons per annum of nicotinamide for use in animal feed.

Structure

NHases are composed of two types of subunits, α and β, which are not related in amino acid sequence. NHases exist as αβ dimers or α₂β₂ tetramers and bind one metal atom per αβ unit. The 3-D structures of a number of NHases have been determined. The α subunit consists of a long extended N-terminal "arm", containing two α-helices, and a C-terminal domain with an unusual four-layered structure (α-β-β-α). The β subunit consists of a long N-terminal loop that wraps around the α subunit, a helical domain that packs with N-terminal domain of the α subunit, and a C-terminal domain consisting of a β-roll and one short helix.

Assembly

An assembly pathway for nitrile hydratase was first proposed when gel filtration experiments found that the complex exists in both αβ and α2β2 forms. In vitro experiments using mass spectrometry further revealed that the α and β subunits first assemble to form the αβ dimer. The dimers can then subsequently interact to form a tetramer.

Mechanism

The metal centre is located in the central cavity at the interface between two subunits. All protein ligands to the metal atom are provided by the α subunit. The protein ligands to the iron are the sidechains of the three cysteine (Cys) residues and two mainchain amide nitrogens. The metal ion is octahedrally coordinated, with the protein ligands at the five vertices of an octahedron. The sixth position, accessible to the active site cleft, is occupied either by NO or by a solvent-exchangeable ligand (hydroxide or water). The two Cys residues coordinated to the metal are post-translationally modified to Cys- sulfinic (Cys-SO₂H) and - sulfenic (Cys-SOH) acids. Quantum chemical studies predicted that the Cys-SOH residue might play a role as either a base (activating a nucleophilic water molecule) or as a nucleophile. Subsequently, the functional role of the SOH center as nucleophile has obtained experimental support.