Aliphatic nitrilases

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]. 

A nitrilase with a preference for fumaronitrile was also obtained by expressing a gene sequenced in a photosynthetic cyanobacterium, Synechocystis sp. PCC6803 [12]. This enzyme, however, also hydrolyzed other substrates (aromatic, aliphatic, dinitriles), albeit at reaction rates two orders of magnitude lower than fumaronitrile. 

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R. D. Fallon, J. E. Gavagan, R. DiCosimo, and M. S. Payne, Purification, cloning, sequendng and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W, Appl. Microbiol. Biotechnol. 2003, 61, 118-122. 

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. 

Levy-Schil, S. Soubrier, F., Crutz-LeCoq, A.M., et al. 1995. Aliphatic nitrilase from a soil isolated Comamonas testosteroni sp. Gene cloning and overexpression, purification and primary structure. Gene, 161 15-20. 

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). 

Investigation of the structure-function relationships in nitrilases was largely based on sequence analyses, homology modeling, and mutational studies, as the crystal structures of nitrile-hydrolyzing enzymes have not been available except for an aliphatic nitrilase from Pyrococcus abyssi [13]. Other crystallized members of the nitrilase superfamily (amidases, N-carbamoyl-D-amino acid amidohydrolases, etc. [9]) shared only low levels of identity with experimentally confirmed nitrilases. 

TABLE 12.3 Catalytic Properties of Purified Aliphatic Nitrilases ... 

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