The Arabidopsis thaliana Isogene NIT4 and Its Orthologs in Tobacco Encode β-Cyano-l-alanine Hydratase/Nitrilase

Abstract

Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NIT4 are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for ␤-cyano-L-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N. tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.

Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana

“…As it is known that (i) the genes for the IAN-converting isoforms 2 and 3 of A. thaliana nitrilase are active specifically during embryo development (NIT2, Vorwerk et al 2001) and early germination (NIT3, Kutz et al 2002) and (ii) amides may be side-products of nitrilase activity (Bartel and Fink 1994;Piotrowski et al 2001), we examined the conversión of IAN by purified, recombinant NIT1, NIT2 and NIT3 (Fig. 5).…”

“…For the expression of nitrilase enzymes, the E. coli strain BL21-CodonPlus (DE3)-RIL (Stratagene, La Jolla, Calif., USA) was used. Purification of the recombinant nitrilases was done as described by Piotrowski et al (2001). The purified nitrilase fraction showed homogeneity of at least 95%, as judged by Coomassie-blue stained SDS-polyacrylamide gels.…”

Transcription Analysis of Arabidopsis Membrane Transporters and Hormone Pathways during Developmental and Induced Leaf Senescence

“…6D). In addition, a nitrilase (AtNIT4) that is thought to play a role in cyanide detoxification during ET biosynthesis (Piotrowski et al, 2001) is up-regulated (Supplemental Fig. 6E).…”

Improved Fitness of Arabidopsis thaliana Nitrilase 2

“…[13] NIT4 occurs in taxonomically quite distinct species and has a different substrate specificity compared to NIT1, NIT2, and NIT3. Whereas the substrate specificity for NIT4 is strongly restricted to b-cyano-l-alanine, [14] the three other isoenzymes show a similar substrate preference, accepting a variety of aromatic and aliphatic nitriles. [15] Besides several scientific literature reports about the selective hydrolysis of diverse nitriles using Arabidopsis thaliana nitrilases, [11,[16][17][18][19] there is also a patent application claiming their industrial potential.…”

“…Based on the wild-type sequence (accession number BAE98592.1), a synthetic gene for a starting clone (NIT2wt) was generated that carried an aminoterminus stabilizing glycine [27] adjacent to the methionine, and a C-terminal His-tag [14] to facilitate protein purification. NIT2wt was used as a template and Mutazyme I from the GeneMorph Random Mutagenesis Kit (Stratagene, Foster City, CA) as a polymerase for the first round of ep PCR.…”