The nitrilase family of CN hydrolysing enzymes - a comparative study

O’Reilly, Catherine; Turner, Peter

doi:10.1046/j.1365-2672.2003.02123.x

Cited by 219 publications

(138 citation statements)

References 74 publications

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“…Taking into account the water molecules, the reaction can be written as 2 (3-cyanoalanine) ϩ 3 (H 2 O) ¡ 1 (aspartate) ϩ 1 (asparagine) ϩ 1 (NH 4 ϩ ). This result is somewhat similar to the 2:1 or 1.36:1 ratio obtained for the nitrile hydratase/nitrilase described in plants and other bacterial cyanoalanine nitrilases (29,41,47). In any case, it is remarkable that this enzyme does not accept as a substrate a compound that is assumed to be a reaction intermediate (i.e., asparagine).…”

Section: Discussionsupporting

confidence: 87%

“…Nitrilases are enzymes catalyzing the hydrolysis of nitriles to the corresponding carboxylic acids plus ammonia (41). These enzymes have received extensive attention because they were shown to have several biotechnological applications in the so-called green chemistry (42), including uses in synthetic processes, such as industrial production of acrylamide and nicotinamide (43).…”

Section: Discussionmentioning

confidence: 99%

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A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

Acera

Carmona

Castillo

et al. 2017

Appl Environ Microbiol

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Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded by nit4, one of the four nitrilase genes detected in the genome of this bacterium, and its expression in Escherichia coli enabled the recombinant strain to fully assimilate 3-cyanoalanine. P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, a nit4 knockout mutant of P. pseudoalcaligenes CECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed in E. coli was purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine, and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that the Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activities. The nit4 gene is located downstream of the cyanide resistance transcriptional unit containing cio1 genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed that nit4 expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster including cio1 and nit4 could be involved both in cyanide resistance and in its assimilation by P. pseudoalcaligenes CECT 5344.IMPORTANCE Cyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold mining and the electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to physicochemical treatment. P. pseudoalcaligenes CECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This paper describes and characterizes an enzyme (Nit4) induced by cyanide that is probably involved in cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway in P. pseudoalcaligenes. This information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

Acera

Carmona

Castillo

et al. 2017

Appl Environ Microbiol

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“…22 Only nitrilases are commercially available, but occasional formation of amide in nitrilase-catalyzed hydolysis of nitriles has been observed. 23 This partial hydrolysis may depend on the structure of the nitrile, as in the case reported for a nitrilasecatalyzed hydrolysis of a β-hydroxynitrile.…”

mentioning

confidence: 99%

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

Meroni¹,

Ciana²,

Meda³

et al. 2009

Arkivoc

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“…Nevertheless, there is evidence that for certain amide forming reactions, nature uses enzymes, which are closely related to nitrilases and which belong evolutionarily to the same subgroup within the nitrilase superfamily as true nitrilases. The best-characterized examples for this type of reactions are the cyanide hydratases fromfungalspeciessuchasFusariumoxysporumandGloeoecercospora Y, which convert cyanide to formamide (20). In addition, it has recently been demonstrated that a certain group of plant-derived nitrilases ("NIT4") preferentially catalyze the transformation of 4-cyanoalanine, which is a typical cyanide detoxification product of plants, to the amide asparagine and not to the expected product aspartate (7,23).…”

Section: Discussionmentioning

confidence: 99%

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

Sosedov

Baum

Bürger

et al. 2010

Appl Environ Microbiol

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The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.

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The nitrilase family of CN hydrolysing enzymes - a comparative study

Cited by 219 publications

References 74 publications

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

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