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Nitrilase enzymes (nitrile aminohydrolase; )
catalyse Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
the
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
of
nitrile In organic chemistry, a nitrile is any organic compound that has a functional group. The name of the compound is composed of a base, which includes the carbon of the , suffixed with "nitrile", so for example is called " propionitrile" (or pr ...
s to
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an Substituent, R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl ...
s and
ammonia Ammonia is an inorganic chemical compound of nitrogen and hydrogen with the chemical formula, formula . A Binary compounds of hydrogen, stable binary hydride and the simplest pnictogen hydride, ammonia is a colourless gas with a distinctive pu ...
, without the formation of "free" amide intermediates. 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 mixture In chemistry, a racemic mixture or racemate () is a mixture that has equal amounts (50:50) of left- and right-handed enantiomers of a chiral molecule or salt. Racemic mixtures are rare in nature, but many compounds are produced industrially as r ...
s. Nitrilase should not be confused with
nitrile hydratase Nitrile hydratases (NHases; ) are mononuclear iron or non-corrinoid cobalt enzymes that catalyse the hydration of diverse nitriles to their corresponding amides: : R-C≡N + → Metal cofactor Nitrile hydratases use Fe(III) or Co(III) at thei ...
(nitrile hydro-lyase; ) 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.


Mechanism

Nitrilase was first discovered in the early 1960s for its ability to catalyze the hydration of a
nitrile In organic chemistry, a nitrile is any organic compound that has a functional group. The name of the compound is composed of a base, which includes the carbon of the , suffixed with "nitrile", so for example is called " propionitrile" (or pr ...
to a
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an Substituent, R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl ...
. Although it was known at the time that nitrilase could operate with wide
substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (aquatic environment), the earthy material that exi ...
specificity in producing the corresponding acid, later studies reported the first NHase (
nitrile hydratase Nitrile hydratases (NHases; ) are mononuclear iron or non-corrinoid cobalt enzymes that catalyse the hydration of diverse nitriles to their corresponding amides: : R-C≡N + → Metal cofactor Nitrile hydratases use Fe(III) or Co(III) at thei ...
) activity exhibited by nitrilase. That is,
amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a chemical compound, compound with the general formula , where R, R', and R″ represent any group, typically organyl functional group, groups or hydrogen at ...
compounds could also be formed via nitrile
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
. Further research has revealed several conditions that promote amide formation, which are outlined below. * 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 and 50 °C. * Electron withdrawing groups at the ⍺-position Below is a list of steps involved in transforming a generic nitrile compound with nitrilase: # The
electrophilic In chemistry, an electrophile is a chemical species that forms bonds with nucleophiles by accepting an electron pair. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are positively charged, have an atom that carr ...
carbon of the nitrile is subject to
nucleophilic In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they a ...
attack by one of the two SH groups on nitrilase. # The thioimidate formed is subsequently hydrolyzed to the acylenzyme and
ammonia Ammonia is an inorganic chemical compound of nitrogen and hydrogen with the chemical formula, formula . A Binary compounds of hydrogen, stable binary hydride and the simplest pnictogen hydride, ammonia is a colourless gas with a distinctive pu ...
is created as a byproduct. # 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

Most nitrilases are made up of a single
polypeptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty ...
ranging from 32 to 45 kDa, and its structure is an ⍺-β-β-⍺ fold. The favored form of the enzyme is a large filament consisting of 6-26 subunits. Nitrilase exploits the Lys- Cys- Glu
catalytic triad A catalytic triad is a set of three coordinated amino acid residues that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, aminoac ...
which is essential for its
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
function and enhancing its performance. The structure of a thermoactive nitrilase from P. abyssi consists of a 2-fold symmetric dimer in which each subunit contains 262 residues. 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. 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 Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidinium, guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) a ...
and
glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
residues. 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. 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 An acid is a molecule or ion capable of either donating a proton (i.e. Hydron, hydrogen cation, H+), known as a Brønsted–Lowry acid–base theory, Brønsted–Lowry acid, or forming a covalent bond with an electron pair, known as a Lewis ...
responsible for proton transfer to the substrate.


Biological Function

Nitrilases have critical roles in plant-microbe interactions for defense,
detoxification Detoxification or detoxication (detox for short) is the physiological or medicinal removal of toxic substances from a living organism, including the human body, which is mainly carried out by the liver. Additionally, it can refer to the period o ...
, nitrogen utilization, and plant hormone synthesis. 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
glycoside In chemistry, a glycoside is a molecule in which a sugar is bound to another functional group via a glycosidic bond. Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. ...
s and
glucosinolate Glucosinolates are natural components of many pungent plants such as mustard, cabbage, and horseradish. The pungency of those plants is due to mustard oils produced from glucosinolates when the plant material is chewed, cut, or otherwise damaged. ...
s. The arylcetonitrile substrates for these particular enzymes consist of phenylpropionitrile and other products that result from glucosinolate metabolism. NIT4 however, belongs to the second group of plant nitrilases and is critical for
cyanide In chemistry, cyanide () is an inorganic chemical compound that contains a functional group. This group, known as the cyano group, consists of a carbon atom triple-bonded to a nitrogen atom. Ionic cyanides contain the cyanide anion . This a ...
detoxification in plants. Moreover, microbes could also potentially utilize nitrilase for detoxifying and assimilating nitriles and cyanide that exist in the plant environment. An example of this is the β-cyano-L-alanine nitrilase by the plant bacterium ''P. fluorescens'' SBW25. 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. 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 In organic chemistry, mandelonitrile is the nitrile of mandelic acid, or the cyanohydrin derivative of benzaldehyde. Small amounts of mandelonitrile occur in the pits of some fruits. Occurrence Mandelonitrile is the aglycone part of the cyano ...
, which serves as a defense against herbivores.


Further reading

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References


External links

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