Nitrile hydratase

From Wikipedia, the free encyclopedia
Jump to: navigation, search
nitrile hydratase
EC number
CAS number 82391-37-5
IntEnz IntEnz view
ExPASy NiceZyme view
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO

In enzymology, nitrile hydratases (NHases; EC are mononuclear iron or non-corrinoid cobalt enzymes that catalyse the hydration of diverse nitriles to their corresponding amides

R-C≡N + H2O → R-C(O)NH2

Metal cofactor[edit]

In biochemistry, cobalt is in general found in a corrin 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 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[edit]

Nitrile hydratase and amidase are two hydrating and hydrolytic enzymes responsible for the sequential metabolism of nitriles 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.[1] 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.[2]

Industrial applications[edit]

NHases have been efficiently used for the industrial production of acrylamide from acrylonitrile 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.


Structure of nitrile hydratase.[3]

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.

Nitrile hydratase, alpha chain
Symbol NHase_alpha
Pfam PF02979
InterPro IPR004232
SCOP 2ahj
Nitrile hydratase beta subunit
Symbol NHase_beta
Pfam PF02211
InterPro IPR003168
SCOP 2ahj


An assembly pathway for nitrile hydratase was first proposed when gel filtration experiments found that the complex exists in both αβ and α2β2 forms. [4] 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. [5]


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)[6] or as a nucleophile.[7] Subsequently, the functional role of the SOH center as nucleophile has obtained experimental support.[8]


  1. ^ Foerstner KU, Doerks T, Muller J, Raes J, Bork P (2008). Hannenhalli, Sridhar, ed. "A nitrile hydratase in the eukaryote Monosiga brevicollis". PLoS ONE 3 (12): e3976. doi:10.1371/journal.pone.0003976. PMC 2603476. PMID 19096720. 
  2. ^ Marron AO, Akam M, Walker G (2012). Stiller, John, ed. "Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups". PLoS ONE 7 (4): e32867. doi:10.1371/journal.pone.0032867. 
  3. ^ Nagashima S, Nakasako M, Dohmae N, et al. (May 1998). "Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms". Nat. Struct. Biol. 5 (5): 347–51. doi:10.1038/nsb0598-347. PMID 9586994. 
  4. ^ Payne, MS; Wu, S; Fallon, RD; Tudor, G; Stieglitz, B; Turner, IM; Nelson, MJ (May 1997). "A stereoselective cobalt-containing nitrile hydratase". Biochemistry 36 (18): 5447-54. doi:10.1021/bi962794t. PMID 9154927. 
  5. ^
    Protein complexes are under evolutionary selection to assemble via ordered pathways  
    Language English
  6. ^ Hopmann, KH; Guo JD, Himo F (2007). "Theoretical Investigation of the First-Shell Mechanism of Nitrile Hydratase". Inorg. Chem. 46 (12). doi:10.1021/ic061894c. 
  7. ^ Hopmann, KH; Himo F (March 2008). "Theoretical Investigation of the Second-Shell Mechanism of Nitrile Hydratase". European Journal of Inorganic Chemistry 2008 (9). doi:10.1002/ejic.200701137. 
  8. ^ Salette, M; Wu R, Sanishvili R, Liu D, Holz RC (2014). "The Active Site Sulfenic Acid Ligand in Nitrile Hydratases can Function as a Nucleophile". JACS. doi:10.1021/ja410462j. 

Further reading[edit]

  • Prasad, S; Bhalla, TC (May 2010). "Nitrile hydratases (NHases): At the interface of academia and industry .". Biotechnology Advances 28 (6): 725–41. doi:10.1016/j.biotechadv.2010.05.020. PMID 20685247. 
  • Rzeznicka, K; Schätzle, S; Böttcher, D; Klein, J; Bornscheuer, UT (Aug 2009). "Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation.". Appl Microbiol Biotechnol 85 (5): 1417–25. doi:10.1007/s00253-009-2153-y. PMID 19662400. 
  • Song, L; Wang, M; Yang, X; Qian, S (Jun 2007). "Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus sp. AJ270.". Biotechnol J 2 (6): 717–24. doi:10.1002/biot.200600215. PMID 17330219. 
  • Miyanaga, A; Fushinobu, S; Ito, K; Shoun, H; Wakagi, T (Jan 2004). "Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.". Eur J Biochem 271 (2): 429–38. doi:10.1046/j.1432-1033.2003.03943.x. PMID 14717710. 
  • Hann, EC; Eisenberg, A; Fager, SK; Perkins, NE; Gallagher, FG; Cooper, SM; Gavagan, JE; Stieglitz, B; Hennessey, SM; DiCosimo, R (October 1999). "5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23.". Bioorg Med Chem 7 (10): 2239–45. doi:10.1016/S0968-0896(99)00157-1. PMID 10579532.