Nitrilase substrate specificity

A nitrilase from the hyperthermophile Pyrococcus abyssi, which exhibits optimal growth at 100 °C, was cloned and overexpressed. Characterization of this nitrilase revealed that it is operational as a dimer (rather than the more common multimeric structure for nitrilases), with optimal pH at 7.4 and optimal apparent activity at 80 °C with Tm (DSC) at 112.7 °C. The substrate specificity of the nitrilase is narrow and it does not accept aromatic nitriles. The nitrilase converts the dinitriles fumaronitrile and malononitrile to their corresponding mononitriles [58],... 

A systematic study of the substrate specificity profile of this nitrilase (Table 8.6) illustrated that arylacetonitriles, including phenylacetonitrile derivatives indole-3-acetonitrile and... 

Table 8.6 Summary of substrate specificity of Pseudomonas putida nitrilase (data is not comprehensive)... 

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... 

Zhu, D., Murkherjee, C., Yang, Y, et al. 2008. A new nitrilase from Bradyrhizopium japonicum USDA 110 Gene cloning, biochemical characterization and substrate specificity. 

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. 

Table 14.3 Substrate specificity of purified nitrilases from filamentous fungi comparison with prokaryotic nitrilases.

A method of efficient induction of fungal nitrilases in wild strains was established and seems to be of wide application throughout this enzyme group. However, heterologous expression of fungal nitrilases remains to be solved in order to facilitate the use of these enzymes in industrial biotechnology. To what extent the desirable properties (broad substrate specificity, thermostability, chemoselectivity and enantioselectivity) can be improved by mutagenesis of these enzymes should also be examined. 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

Therefore, in order to obtain the carboxylic acid as the final product, it was proposed to transform the amide using an amidase. The reactions can proceed in a single reactor or in two reactors connected in series. The utility of this method was exemplified by the hydrolysis of 4-cyanopyridine into isonicotinic acid [56], an intermediate in the synthesis of the tuberculostatic drug isoniazid. Nitrilases from Aspergillus niger or Fusarium solani and an amidase from R. erythropolis were chosen for this process, as they exhibited compatible substrate specificities. If the cascade reactions were carried out in two separate reactors, a crude extract from R. erythropolis A4 cells could serve as the amidase source. In a one-pot reaction, the NHase present in this extract would compete with the nitrilase and increase... 

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... 

A nitrilase sequenced in the archaeon P. abyssi GE5 was expressed in E. coli to give a highly thermostable enzyme (with an enzyme half-life of 25, 9, and 6h at 70, 80, and 90 °C, respectively [14]). This enzyme is the only experimentally confirmed nitrilase to have been crystallized [13]. The low identity of this enzyme to other characterized nitrilases makes its usefulness for homology modeling low. This enzyme also differs from most other characterized nitrilases in its substrate specificity, its preferential substrates being fumaronitrile and malononitrile [14]. 

Nitrilase. II. Substrate specificity and possible mode of action. Arch. Biochem. Biophys., 107, 62-68. 

Nitrilases are widespread in plants, fungi, archaea, and bacteria [23-25], possibly owing to their central role in metabolizing organocyanides as carbon and/ or nitrogen sources [26]. The enzyme family can be divided into three groups according to substrate specificity [27] ... 

Nitrile hydratases (NHases) exhibit broader substrate specificities than nitrilases, and they are more tolerant of sterically demanding substrates. In contrast, NHases mainly exhibit lower enantioselectivities than nitrilases, although biotransformations of a few specific nitriles by NHase proceeded with excellent enantioselectivities [5,6]. Low NHase enantioselectivity, however, can be compensated for by using enantiose-lective amidases in the next step. NHases are usually less thermostable than nitrilases, but a few of them are resistant to increased temperatures or organic solvent concentrations [8, 9]. The majority of characterized NHases are Fe or Co type, except for a new NHase with three metal ions (Co, Cu, Zn) reported in Rhodococcus jostii [85]. 

Nitrilases in plants are comprised of two subtypes with different biological functions. The first subtype involves nitrilases exhibiting mostly over 70% identity levels to the biochemically characterized nitrilases 1-3 from A. thaliana, which act on substrates such as 3-phenylpropionitrile, allylcyanide, or indole-3-acetonitrile. They seem to be specific for Brassicaceae, which is in accordance with their preference for the nitriles endogeneous in this family or structurally similar to them [22]. They are able to produce plant auxins such as indole-3-acetic acid but the role of this pathway in vivo is not fully understood [21]. 

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