Cyanide hydratase

Cyanide hydratase, also known as formamide hydrolyase, has been reported to hydrolyze cyanide to formamide [57,58], Cyanide hydratase is produced by a variety of fungi and is inducible upon preexposure of the fungi to low concentrations of cyanide. It was noted that cyanide hydratase was more stable when immobilized and that the enzyme from Gloeocerco-... 

Cyanide hydratase and cyanide dihydratase belongs to the nitrilase branch of nitrilase superfamily, using HCN as the only efficient substrate and producing amide and acid products, respectively. Microorganisms appear in fact to have evolved separate metabolic pathways for the hydrolysis of inorganic cyanide. Thus, most nitrilases (as well as nitrile hydratases) till now investigated do not display activity... 

Gloeoecercospora sorghi Cyanide hydratase Wang and Van Etten (1992)... 

Clnness, M.J., Tnmer, P.D. Clements, E., Brown, D.T., and O Reilly, C. 1993. Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chyl gene. Journal of General Microbiology, 139 1807-15. 

Nolan, L.M., Harnedy, P.A., Tinner, R, et al. 2003. The cyanide hydratase enzyme of Eusarium lateritium also has nitrilase activity. EEMS Microbiology letters, 221 161-5. 

Novo, C., Tata, R., Clemente, A., et al. 1995. Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys is predicted to be the active site nucleophile of the catalytic mechanism. EEBS letters, 367 275-9. 

Sexton, A.C. and Howlett, B.J. 2000. Characterization of a cyanide hydratase gene in the phy-topaihog dicfxmgvis Leptosphaeria maculans. Gene, 263 463-470. 

Wang, R, Matthews, D., and Van Etten, H. 1992. Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi. Archives in Biochemistry and Biophysics, 298 569-75. 

Wang, P. and Van Etten, H.D. 1992. Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi. Biochemical and Biophysical Research Communications, 187 1048-54. 

On the basis of sequence data and biochemical characterization, this chapter wiU compare the structure and enzymatic properties of nitrilases from filamentous fungi with those of prokaryotic and plant nitrilases as well as other related enzymes like cyanide hydratases and cyanide dihydratases. The biotechnological potential of fungal nitrilases will be evaluated. 

Table 14.1 Subunit and holoenzyme molecular masses of nitrilases from filamentous fungi comparison with selected prokaryotic nitrilases, cyanide hydratases and cyanide dihydratase.

The nitrilase from F. solani IMI196840 also differs from other fungal nitrilases in terms of its value for benzonitrile (0.039 mM) [10], which is markedly lower than those of other fungal nitrilases (0.2-1.5mM) [11, 14, 15]. In general, even higher fCm-values, that is in the range of 2.6-12 mM for wild-type enzymes and 5.9-90 mM for hexahistidine-tagged enzymes, have been determined for cyanide hydratases/dihydratases [17]. 

Enzymes catalyzing the hydrolysis of cyanide had already been identified earlier. Here too, two different types can be distinguished. The enzyme cyanide hydratase [formamide hydrolase (E. C. 4.2.1.66)] can be found in various fungi1311. It catalyzes the hydration of cyanide to formamide ... 

Nitrilases are classified into branch 1 of the nitrilase superfamily, which is comprised of enzymes acting on various nonpeptide CN bonds [15]. All the proteins of this superfamily are characterized by a conserved catalytic triade (glu, lys, cys) and an additional conserved glu residue that seems to participate in the reaction mechanism [2]. Members of class 1 transform the CN bonds in nitriles and cyanides. The enzymes in which these activities were confirmed share in some cases levels of aa sequence identity as low as about 20%. This sequence diversity is reflected in different substrate specificities and different reaction products (carboxylic acids, amides) in various subtypes of these enzymes (aromatic nitrilases, aliphatic nitrilases, arylacetonitrilases, cyanide hydratases, cyanide dihydratases). 

Table 12.1 Characterized nitrilase and cyanide hydratases predicted from sequence data.

Cyanide hydratase Aspergillus nidulans FGSC A4 tpe CBF78050.1 [5]... 

There are many examples of nitrilase-catalyzed reactions in which amides form a considerable amount of the reaction products, such as the transformations of acrylonitrile analogs and a-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis thaliana [17], the conversion of p-cyano-L-alanine into a mixture of L-asparagine and L-aspartic acid by nitrilase 4 from the same organism [18] or the transformations of mandelonitrile by nitrilase from Pseudomonas jhiorescens [19] or some fungi [8], Moreover, formamide is the only product of the cyanide transformation by cyanide hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although it is closely related to nitrilases, as far as its aa sequence and reaction mechanism are concerned [3]. 

Cyanide hydratases are more closely related to each other, with over 50% identities. It was hypothesized [20] that their ancestor genes were acquired by fungi from bacteria via horizontal gene transfer, which was followed by neofunctualization of... 

The well-known classification of nitrilases into different substrate specificity sub-types [2] was demonstrated to be in partial correlation with their aa sequence similarities. A prediction of substrate specificities in putative nitrilases could be made by grouping similar sequences. For instance, four probable arylacetonitri-lases were selected because of their high similarity (over 50%) to the biochemically characterized enzyme from Neurospora crassa, and their expected substrate specificities were confirmed [6]. Using this approach, it also seems possible to predict cyanide hydratases [5,6]. Aromatic nitrilases are not as easy to predict Members of... 

The region downstream of the catalytic Cys also allows nitrilases and cyanide hydratases to be distinguished relatively successfully. The experimentally confirmed cyanide hydratases contain an Asn residue at position 3 downstream of the catalytic Cys [5, 6], whereas most of the characterized nitrilases carry a His at this position except for a few enzymes (from A. thaliana and Klebsiella ozaenae with an Asn at the corresponding site [3]). 

The spectrum of known nitrilases and cyanide hydratases currently consists of a significant part of enzymes obtained by genome mining. These enzymes were, in many cases, helpful to the improvement of processes employing the hydrolysis of... 

Further, E. coli cells expressing the cyanide hydratases genes from A. niger and P, chrysogenum were found to exhibit activities for both HCN and some nitrile compounds, preferably 2-cyanopyridine (1-3.6% relative activity compared to HCN) [6]. Dual nitrilase/cyanide hydratase activities were also described for the enzymes in Fusarium oxysporum and Fusarium lateritium [36, 37]. It is possible that this dual activity is a general feature of cyanide hydratases but has largely gone unnoticed. 

P-Cyanoalanine can be transformed into y-glutamyl f -cyanoalanine by y-glutamyl transferase. Other enzymes involved in cyanide catabolism are cyanide hydratase and rhodanase. Cyanide hydratase, found in many fungi, catalyses the hydration of cyanide to formamide (H2N-CH=0). Rhodanase found in bacteria, plants and animals catalyses the conversion of cyanide into thiocyanate (R-S C N), which is also known as rhodanide. 

Cyanopyridine (precursor of picolinic acid, intermediate of pharmaceuticals) is transformed by aromatic nitrilases at lower relative rates compared to arylace-tonitrilases. Surprisingly, the cyanide hydratase from A. niger KIO exhibited the highest specific activity for this substrate among the enzymes examined [18, 72]. This enzyme, as well as other cyanide hydratases (from the Fusarium genus), accepted a number of nitrile substrates, albeit at much lower relative rates than HCN [3, 72]. 

Rin gelov, A., Kaplan, O., Vesela, A.B., Chmatal, M., Kfenkova, A., Plfhal, O., PasquareUi, R, Cantarella, M., and Martmkova, L. (2014). Cyanide hydratase from Aspergillus niger KIO Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation. Process Biochemistry, 49,445-450. 

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