Aromatic nitrilases

Aromatic nitrilases were first described in Rhodococci in the 1980s [3]. The enzyme from Rhodococcus rhodochrous J1 is one of the best characterized enzymes of this type [61]. A similar enzyme with a moderate level of identity (61.6%) was recently described in . putida [62]. 

Aromatic nitrilases of fungal origin have also been known for decades [3], but their amino acid (AA) sequences were only determined later by MS analysis [63-66]. These sequences were similar to those of enz5unes from Gibberella monilifotmis and Gibberella intermedia, which were then produced in E. coli [43,67] and confirmed as aromatic nitrilases. 

2-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]. 

The majority of aromatic nitrilases hydrolyze not only (hetero) aromatic but also aliphatic saturated or unsaturated nitriles (Table 12.2). The ability of some of the enzymes to hydrolyze acrylonitrile has an industrial impact, as it enables the synthesis of acrylic acid (a polymer building block), the elimination of acrylonitrile from wastewaters [73], or the construction of acrylonitrile biosensors [74]. The nitrilase from R. rhodochrous J1 required activation by ammonium sulfate prior to use for the transformation of acrylonitrile. This activation resided in a rearrangement of the enzyme s quaternary structure, resulting in an increase in molecular weight from 

12 NITRILE-CONVERTING ENZYMES AND THEIR SYNTHETIC APPLICATIONS 

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Nitrilase was initially discovered in plants as an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) [74,75], Recently, four genes of nitrilases (belonging to arylacetonitrilase) involved in the IAA biosynthesis have been cloned and characterized from Arabidopsis thaliana [76-78], After the discovery of the plant nitrilase in 1964, various nitrilases were purified and characterized [41], Nitrilases are roughly classified into three major categories according to substrate specificity (i) aromatic nitrilase, which acts on aromatic or heterocyclic nitriles (ii) aliphatic nitrilase, which acts on aliphatic nitriles (iii) arylacetonitrilase, which acts on arylacetonitriles. These three types... 

All nitrilases belonging to aromatic nitrilase and arylacetonitrilase are susceptible to SH-reagents, except the R. rhodochrous K22 aliphatic nitrilase [81]. Analysis by site-directed mutagenesis for our three nitrilases from R. rhodochrous J1 [83], R. rhodochrous K22 [84] and Alcaligenes faecalis JM3 [85] revealed that a unique cysteine that is conserved at the corresponding position in each nitrilase is essential for the catalytic activity (Fig. 7). All nitrilases whose structural genes are cloned have a similar amino acid sequence nitrilase forms a superfamily. 

All known fungal nitrilases exhibit high relative activities towards benzonitrile and its m- and p-substituted derivatives. Therefore, according to the nitrilase classification [46] they belong to aromatic nitrilases. However, almost all fungal nitrilases also hydrolyze aUphatic nitriles, albeit at lower relative rates. 

The substrate specificities of all four biochemically characterized fungal nitrilases and two enzymes from rhodococci (an aromatic and an aUphatic nitrilase) are compared in Table 14.3. Only a few nitriles were examined as substrates of aU these enzymes. Moreover, the comparison of substrate specificity is difficult in some cases as different activity assays have been used for different enzymes. For instance, the determination of activity by ammonia measurement did not reflect potential amide formation. Nevertheless, common patterns can be found in substrate preferences of aromatic nitrilases. 

Among aliphatic nitriles, unbranched saturatured or unsaturated compounds with a medium chain length (for instance, propionitrile, butyronitrile, hexaneni-trile, and acrylonitrile) are hydrolyzed with the highest relative rate (about 20-35% of that for benzonitrile) by aromatic nitrilases. Branched nitriles are in general poor substrates of these enzymes but, for instance, isobutyronitrile is one of the best substrates of the aliphatic nitrilase from R. rhodochrous K22 [28]. 

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). 

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... 

Aromatic nitrilases, which mainly act on (hetero-)aromatic nitriles, for example, benzonitrile... 

TABLE 12.2 Catalytic Properties of Purified Aromatic Nitrilases ... 

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