Nhase

In enzymology, nitrile hydratases (NHases; EC 4.2.1.84) involved in microbial nitrile assimilation can be roughly classified into two groups according to the metal involved: mononuclear iron and non-corrinoid cobalt enzymes, which catalyze the hydration of diverse nitriles to their corresponding amides that is then converted into carboxylic acid by an amidase. NHase holds great promise as catalysts in organic chemical processing because they can convert nitriles to the corresponding higher-value amides under mild conditions and has become one of the most important industrial enzymes since it is able to accumulate extraordinarily high concentration of amides in a strikingly fast manner. NHase and amidase as two hydrating and hydrolytic enzymes are responsible for the sequential metabolism of nitriles in bacteria, which are able to utilize nitriles as their sole source of nitrogen and carbon, and they could in concert function as an alternative to nitrilase that causes nitrile hydrolysis without formation of an intermediate primary amide.

Sources

A large number of NHases have been detected in a range of microorganisms. The collective of known mesophilic NHase-producing microorganisms is dominated by genus Rhodococcus, and only five thermophilic NHase-producing organisms have far been reported. Various NHase subgroups exhibit significant differences in origin, stability, cofactor requirements, and catalytic characteristics, whereas there is a high degree of similarity in terms of size and protein sequence between these enzymes. A sequence in genome of the choanoflagellate Monosiga brevicollis could encode for a NHase, and the gene of Monosiga brevicollis consisted of both α and β subunits that are fused into a single gene. Similar NHase genes composed of a fusion of the α and β subunits have also been identified in several eukaryotic supergroups, suggesting that such NHases are present in the last common ancestor of all eukaryotes.

Molecular Structure

NHases consist of α and β subunits that are not related in amino acid sequence, exist in forms of αβ dimers or α₂β₂ tetramers and bind one metal atom per αβ unit. The determination of 3D structures of numerous NHases indicates that the α subunit is composed of a long extended N-terminal arm, including two α-helices, and a C-terminal domain with an unusual four-layered structure. The β subunit covers a long N-terminal loop wraping around α subunit, a helical domain packing with N-terminal domain of α subunit, and a C-terminal domain with a β-roll and one short helix. It is found that NHase exists in both αβ and α₂β₂ forms, and in vitro mass spectrometry experiments further reveals that α and β subunits could first assemble to form the αβ dimer, which can subsequently interact to form a tetramer.

Metal Cofactor

In biochemistry, cobalt generally exists in a corrin ring, and NHase is one of the rare types of enzymes that utilize cobalt in a non-corrinoid manner. Although a cobalt permease that transports cobalt across the cell membrane has been identified, the mechanism through which the cobalt is transported to NHase without triggering toxicity is still unclear. The presence of the amino acid sequence VCTLC signifies a Co-centered NHase and the existence of VCSLC manifests a Fe-centered NHase. The identity of the metal in the active site of a NHase can be predicted by analyzing the sequence data of α subunit in the region where the metal is attached.

Mechanism

The metal center is located in the central cavity at the interface between two subunits, and all protein ligands to the metal atom are provided by α subunit. Two carboxamido nitrogens, one cysteine sulfur, and two sulfurs from cysteine-sulfenic (Cys-SO) and cysteine-sulfinic (Cys-SO₂) acid moiety respectively constitute the donor setting around the metal center of NHase. The metal ion is octahedrally coordinated, with the protein ligands at the five vertices of the octahedron. The sixth position is occupied either by NO or a solvent-exchangeable ligand (hydroxide or water). Binding of NO at the sixth site of iron(III) center could modulate the activity of the enzyme, while it is also suggested that a metal-bound hydroxide could make contribution to the hydration of nitriles. The two Cys residues coordinated to the metal are translationally modified into Cys-sulfinic (Cys-SO₂) and Cys-sulfenic (Cys-SO) acids. Quantum chemical studies have predicted that Cys-SO residue might play as either a base to activate a nucleophilic water molecule or as a nucleophile. Structural and spectroscopic studies on iron(III) and cobalt(III) complexes have demonstrated that the unusual coordination structure of the Metal(III) site could raise its potential, shut off any redox activity, allow the binding of NO at the sixth site and the oxidative modification of the bound S donors, and also bring the pKa of metal-bound water close to 7.

Industrial Applications

NHase has long been efficiently employed in the industrial production of acrylamide from acrylonitrile on a scale of 600 000 tons per annum, which is the first successful example of a biotransformation process for the manufacture of a chemical commodity. NHases have also been used for the removal of nitriles from wastewater. NHases possessing nitric oxide (NO) bound to the iron center are intrinsically photosensitive and can be activated through photodissociation. Nicotinamide is produced industrially by hydrolyzing 3-cyanopyridine under the catalysis of the nitrile hydratase from Rhodococcus rhodochrous J1, simultaneously generating 3500 tons per annum of nicotinamide for application in animal feed.

Reference

1. Yamada H, Kobayashi M. Nitrile hydratase and its application to industrial production of acrylamide. Biosci Biotechnol Biochem, 1996, 60(9):1391-400.