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nitrilase 1
Other data
LocusChr. 1 pter-qter

Nitrilase enzymes (nitrile aminohydrolase; EC catalyse the hydrolysis of nitriles to carboxylic acids and ammonia, without the formation of "free" amide intermediates.[1] Nitrilases are involved in natural product biosynthesis and post translational modifications in plants, animals, fungi and certain prokaryotes. Nitrilases can also be used as catalysts in preparative organic chemistry. Among others, nitrilases have been used for the resolution of racemic mixtures. Nitrilase should not be confused with nitrile hydratase (nitrile hydro-lyase; EC which hydrolyses nitriles to amides. Nitrile hydratases are almost invariably co-expressed with an amidase, which converts the amide to the carboxylic acid. Consequently, it can sometimes be difficult to distinguish nitrilase activity from nitrile hydratase plus amidase activity.


Nitrilase was first discovered in the early 1960s for its ability to catalyze the hydration of a nitrile to a carboxylic acid.[2] Although it was known at the time that nitrilase could operate with wide substrate specificity in producing the corresponding acid, later studies reported the first NHase (nitrile hydratase) activity exhibited by nitrilase.[3][4] That is, amide compounds could also be formed via nitrile hydrolysis. Further research has revealed several conditions that promote amide formation, which are outlined below.[4]

  • Early release of the enzyme-bound substrate after the first water hydrolysis followed by delayed addition of the second water
  • Low temperature and increased pH conditions. For bioconversions by nitrilase for most bacteria and fungi, the optimal pH range is between 7.0-8.0 and the optimal temperature range is between 30-50℃.
  • Electron withdrawing groups at the ⍺-position

Below is a list of steps involved in transforming a generic nitrile compound with nitrilase:[4]

  1. The electrophilic carbon of the nitrile is subject to nucleophilic attack by one of the two SH groups on nitrilase.
  2. The thioimidate formed is subsequently hydrolyzed to the acylenzyme and ammonia is created as a byproduct.
  3. The acylenzyme can undergo one of two pathways depending on the conditions highlighted above:
    • Further hydrolyzation of the acylenzyme with water produces the carboxylic acid and the regenerated enzyme.
    • The acylenzyme is hydrolyzed by ammonia, displacing the enzyme and forming the amide product.


File:Nitrilase Active Site.png
The active site of a thermoactive nitrilase from Pyrococcus abyssi, detailing the Lys-Cys-Glu catalytic triad responsible for cleaving C-N bonds. Unfortunately, attempts to crystallize the enzyme with either fumaro- or malononitrile have been ineffective so the binding motif remains unknown.[1]

Most nitrilases are made up of a single polypeptide ranging from 32-45 kDa,[7] and its structure is an ⍺-β-β-⍺ fold.[4] The favored form of the enzyme is a large aggregate consisting of 6-26 subunits.[7] Nitrilase exploits the lys-cys-glu catalytic triad which is essential for its active site function and enhancing its performance.[4][7]

The structure of a thermoactive nitrilase from P. abyssi consists of a 2-fold symmetric dimer in which each subunit contains 262 residues.[8][9] Similar to other nitrilases in the nitrilase family, each subunit has an ⍺-β-β-⍺ sandwich fold; when the two subunits come together and interact, the protein forms a ‘super-sandwich’ (⍺-β-β-⍺-⍺-β-β-⍺) structure.[1] In order to dimerize, the C-terminals of each subunit extend out from the core and interact with each other, and this is largely made possible by the salt bridges formed between arginine and glutamate residues.[1]

Although the exact binding mechanism to the nitrile substrate still remains unknown, by drawing comparisons between the sequence and structure with other nitrilases, the catalytic triad was determined to consist of Glu 42, Lys 113, and Cys 146.[1][4][7] With the aid of protein modeling programs, Glu 42 was observed to be the catalytic base in activating the nucleophile (Cys 146) based on the relatively short distance between the O in Glu and S in Cys. Likewise, Lys 113 was inferred to be the catalytic acid responsible for proton transfer to the substrate.[8][10]

Biological Function

Nitrilases have critical roles in plant-microbe interactions for defense, detoxification, nitrogen utilization, and plant hormone synthesis.[11] In plants, there are two distinguishable groups in regard to substrate specificity: those with high hydrolytic activity towards arylacetonitriles and those with high activity towards β-cyano-L-alanine. N121, 2, and 3 of the A. thaliana species are examples of the first group of plant nitrilases (arylacetonitrilases) which hydrolyze the nitriles produced during the synthesis or degradation of cyanogenic glycosides and glucosinolates. The arylcetonitrile substrates for these particular enzymes consist of phenylpropionitrile and other products that result from glucosinolate metabolism.[11][12] N1T4 however, belongs to the second group of plant nitrilases and is critical for cyanide detoxification in plants.[3][11][13]

Moreover, microbes could also potentially utilize nitrilase for detoxifying and assimilating nitriles and cyanide that exist in the plant environment.[11] An example of this is the β-cyano-L-alanine nitrilase by the plant bacterium P. fluorescens SBW25.[14] Although it is unknown whether this plant bacterium encounters toxic levels of β-cyano-ʟ-alanine in natural settings, nitrilase activity has been observed in cyanogenic plants; thus, it seems that the nitrilase serves as a predominant mechanism for detoxifying cyanide instead of β-cyano-ʟ-alanine.[11][14] Other bacterial applications of nitrilases produced by plant-associated microorganisms include the degradation of plant nitriles for a carbon and nitrogen source. P. fluorescens EBC191 hydrolyzes many arylacetonitriles, namely mandelonitrile, which serves as a defense against herbivores.[11][15][16]

Further reading

  • Winkler M, Glieder A, Klempier N (March 2006). "Enzyme stabilizer DTT catalyzes nitrilase analogue hydrolysis of nitriles". Chemical Communications (12): 1298–300. doi:10.1039/B516937B. PMID 16538253.


  1. 1.0 1.1 1.2 1.3 1.4 Raczynska JE, Vorgias CE, Antranikian G, Rypniewski W (February 2011). "Crystallographic analysis of a thermoactive nitrilase". Journal of Structural Biology. 173 (2): 294–302. doi:10.1016/j.jsb.2010.11.017. PMID 21095228.
  2. Thimann KV, Mahadevan S (April 1964). "Nitrilase. I. Occurrence, Preparation, and General Properties of the Enzyme". Archives of Biochemistry and Biophysics. 105: 133–41. doi:10.1016/0003-9861(64)90244-9. PMID 14165487.
  3. 3.0 3.1 Piotrowski M, Schönfelder S, Weiler EW (January 2001). "The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase". The Journal of Biological Chemistry. 276 (4): 2616–21. doi:10.1074/jbc.M007890200. PMID 11060302.
  4. 4.0 4.1 4.2 4.3 4.4 4.5 Gong JS, Lu ZM, Li H, Shi JS, Zhou ZM, Xu ZH (October 2012). "Nitrilases in nitrile biocatalysis: recent progress and forthcoming research". Microbial Cell Factories. 11: 142. doi:10.1186/1475-2859-11-142. PMC 3537687. PMID 23106943.
  5. Heinemann U, Engels D, Bürger S, Kiziak C, Mattes R, Stolz A (2003). "Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein". Applied and Environmental Microbiology. 69 (8): 4359–66. doi:10.1128/AEM.69.8.4359-4366.2003. PMC 169084. PMID 12902216.
  6. Pace HC, Brenner C (2001-01-01). "The nitrilase superfamily: classification, structure and function". Genome Biology. 2 (1): REVIEWS0001. doi:10.1186/gb-2001-2-1-reviews0001. PMC 150437. PMID 11380987.
  7. 7.0 7.1 7.2 7.3 O'Reilly C, Turner PD (2003). "The nitrilase family of CN hydrolysing enzymes - a comparative study". Journal of Applied Microbiology. 95 (6): 1161–74. doi:10.1046/j.1365-2672.2003.02123.x. PMID 14632988.
  8. 8.0 8.1 Nakai T, Hasegawa T, Yamashita E, Yamamoto M, Kumasaka T, Ueki T, Nanba H, Ikenaka Y, Takahashi S, Sato M, Tsukihara T (July 2000). "Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases". Structure. 8 (7): 729–37. doi:10.1016/s0969-2126(00)00160-x. PMID 10903946.
  9. Pace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, Croce CM, Brenner C (2017-07-27). "Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers". Current Biology. 10 (15): 907–17. doi:10.1016/s0960-9822(00)00621-7. PMID 10959838.
  10. Yeates TO (1997-01-01). "Detecting and overcoming crystal twinning". Methods in Enzymology. 276: 344–58. doi:10.1016/s0076-6879(97)76068-3. PMID 9048378.
  11. 11.0 11.1 11.2 11.3 11.4 11.5 Howden AJ, Preston GM (July 2009). "Nitrilase enzymes and their role in plant-microbe interactions". Microbial Biotechnology. 2 (4): 441–51. doi:10.1111/j.1751-7915.2009.00111.x. PMC 3815905. PMID 21255276.
  12. Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Müller A, Weiler EW, Piotrowski M (March 2001). "Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster". Planta. 212 (4): 508–16. doi:10.1007/s004250000420. PMID 11525507.
  13. Piotrowski M (November 2008). "Primary or secondary? Versatile nitrilases in plant metabolism". Phytochemistry. 69 (15): 2655–67. doi:10.1016/j.phytochem.2008.08.020. PMID 18842274.
  14. 14.0 14.1 Howden AJ, Harrison CJ, Preston GM (January 2009). "A conserved mechanism for nitrile metabolism in bacteria and plants". The Plant Journal. 57 (2): 243–53. doi:10.1111/j.1365-313X.2008.03682.x. PMID 18786181.
  15. Kiziak C, Conradt D, Stolz A, Mattes R, Klein J (November 2005). "Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme". Microbiology. 151 (Pt 11): 3639–48. doi:10.1099/mic.0.28246-0. PMID 16272385.
  16. Legras JL, Chuzel G, Arnaud A, Galzy P (June 1990). "Natural nitriles and their metabolism". World Journal of Microbiology & Biotechnology. 6 (2): 83–108. doi:10.1007/BF01200927. PMID 24429979.

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