Nitrilase

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nitrilase 1
3IVZ.pdb.png
Identifiers
SymbolNIT1
NCBI gene 4817
HGNC 7828
OMIM 604618
PDB 3IVZ
RefSeq NM_005600
UniProt Q86X76
Other data
Locus Chr. 1 pter-qter
Search for
Structures Swiss-model
Domains InterPro

Nitrilase enzymes (nitrile aminohydrolase; EC 3.5.5.1) 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 4.2.1.84) 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.

Contents

Mechanism

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]

The conversion of a general nitrile to either an amide or carboxylic acid is facilitated by nitrilase. Nitrilase.jpg
The conversion of a general nitrile to either an amide or carboxylic acid is facilitated by nitrilase.

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.

Structure

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. 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.

Most nitrilases are made up of a single polypeptide ranging from 32 to 45 kDa, [7] and its structure is an ⍺-β-β-⍺ fold. [4] The favored form of the enzyme is a large filament 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. [6] 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. [6]

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. [6] [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. NIT1, 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] NIT4 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

Related Research Articles

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β-Galactosidase Family of glycoside hydrolase enzymes

β-Galactosidase is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.

In organic chemistry, a nitrile is any organic compound that has a −C≡N functional group. The name of the compound is composed of a base, which includes the carbon of the −C≡N, suffixed with "nitrile", so for example CH3CH2C≡N is called "propionitrile". The prefix cyano- is used interchangeably with the term nitrile in industrial literature. Nitriles are found in many useful compounds, including methyl cyanoacrylate, used in super glue, and nitrile rubber, a nitrile-containing polymer used in latex-free laboratory and medical gloves. Nitrile rubber is also widely used as automotive and other seals since it is resistant to fuels and oils. Organic compounds containing multiple nitrile groups are known as cyanocarbons.

<span class="mw-page-title-main">Aspartate transaminase</span> Enzyme involved in amino acid metabolism

Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or (serum) glutamic oxaloacetic transaminase, is a pyridoxal phosphate (PLP)-dependent transaminase enzyme that was first described by Arthur Karmen and colleagues in 1954. AST catalyzes the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, red blood cells and gall bladder. Serum AST level, serum ALT level, and their ratio are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.

β-Glucuronidase Class of enzymes

β-Glucuronidases are members of the glycosidase family of enzymes that catalyze breakdown of complex carbohydrates. Human β-glucuronidase is a type of glucuronidase that catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides such as heparan sulfate. Human β-glucuronidase is located in the lysosome. In the gut, brush border β-glucuronidase converts conjugated bilirubin to the unconjugated form for reabsorption. β-Glucuronidase is also present in breast milk, which contributes to neonatal jaundice. The protein is encoded by the GUSB gene in humans and by the uidA gene in bacteria.

<span class="mw-page-title-main">Catalytic triad</span> Set of three coordinated amino acids

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.

<span class="mw-page-title-main">Carboxypeptidase</span>

A carboxypeptidase is a protease enzyme that hydrolyzes (cleaves) a peptide bond at the carboxy-terminal (C-terminal) end of a protein or peptide. This is in contrast to an aminopeptidases, which cleave peptide bonds at the N-terminus of proteins. Humans, animals, bacteria and plants contain several types of carboxypeptidases that have diverse functions ranging from catabolism to protein maturation. At least two mechanisms have been discussed.

<span class="mw-page-title-main">Hexosaminidase</span> Class of enzymes

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Stephen aldehyde synthesis, a named reaction in chemistry, was invented by Henry Stephen (OBE/MBE). This reaction involves the preparation of aldehydes (R-CHO) from nitriles (R-CN) using tin(II) chloride (SnCl2), hydrochloric acid (HCl) and quenching the resulting iminium salt ([R-CH=NH2]+Cl−) with water (H2O). During the synthesis, ammonium chloride is also produced.

<span class="mw-page-title-main">Myrosinase</span> Class of enzymes

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<span class="mw-page-title-main">Microsomal epoxide hydrolase</span>

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<span class="mw-page-title-main">Cutinase</span> Class of enzymes

The enzyme cutinase is a member of the hydrolase family. It catalyzes the following reaction:

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<span class="mw-page-title-main">Amidase</span>

In enzymology, an amidase (EC 3.5.1.4, acylamidase, acylase (misleading), amidohydrolase (ambiguous), deaminase (ambiguous), fatty acylamidase, N-acetylaminohydrolase (ambiguous)) is an enzyme that catalyzes the hydrolysis of an amide. In this way, the two substrates of this enzyme are an amide and H2O, whereas its two products are monocarboxylate and NH3.

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<span class="mw-page-title-main">Omega-amidase</span>

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<span class="mw-page-title-main">Dipeptidase 1</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Nicotinonitrile</span> Chemical compound

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:

References

  1. 1 2 Pace HC, Brenner C (2001). "The nitrilase superfamily: classification, structure and function". Genome Biology. 2 (1): REVIEWS0001. doi: 10.1186/gb-2001-2-1-reviews0001 . PMC   150437 . PMID   11380987.
  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. 1 2 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. 1 2 3 4 5 6 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. Bibcode:2003ApEnM..69.4359H. doi:10.1128/AEM.69.8.4359-4366.2003. PMC   169084 . PMID   12902216.
  6. 1 2 3 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.
  7. 1 2 3 4 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. S2CID   8278739.
  8. 1 2 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. S2CID   4644034.
  10. ↑ Yeates TO (1997-01-01). "Detecting and overcoming crystal twinning". Macromolecular Crystallography Part A. Methods in Enzymology. Vol. 276. pp. 344–58. doi:10.1016/s0076-6879(97)76068-3. ISBN   978-0-12-182177-7. PMID   9048378.
  11. 1 2 3 4 5 6 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. Bibcode:2001Plant.212..508V. doi:10.1007/s004250000420. PMID   11525507. S2CID   25573914.
  13. ↑ Piotrowski M (November 2008). "Primary or secondary? Versatile nitrilases in plant metabolism". Phytochemistry. 69 (15): 2655–67. Bibcode:2008PChem..69.2655P. doi:10.1016/j.phytochem.2008.08.020. PMID   18842274.
  14. 1 2 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. S2CID   5463965.
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