Disease relevance of NIT1

• Translational fusions placing the nit1 full-length promoter and a series of 5'-deletion fragments in front of the uidA gene encoding beta-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum [1].
• From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene [2].
• The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1 [3].

High impact information on NIT1

• Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene [4].
• The nitrilase genes encode very similar proteins that hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and three of the four genes are tandemly arranged on chromosome III [5].
• In each case, indole-3-acetonitrile is the immediate precursor, which is converted into indole-3-acetic acid through the action of nitrilase (nitrile aminohydrolase, EC 3.5.5.1) [6].
• The genome of A. thaliana contains two nitrilase genes [6].
• Nitrilase I is soluble and is expressed throughout development, but at a very low level during the fruiting stage, while nitrilase II is tightly associated with the plasma membrane, is barely detectable in young rosettes, but is strongly expressed during bolting, flowering, and especially fruit development [6].

Biological context of NIT1

• Nitrilase (nitrile aminohydrolase, EC 3.5.5.1) catalyzes the hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA) [7].
• A full-length genomic clone encoding the complete cluster of the At nitrilases 1 to 3 (NIT1-3), including the respective promoter regions, has been isolated and the NIT1 isoform has been sequenced [8].
• Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana [1].
• The coding region (nit1) spans about 2.3 kb and is composed of five exons separated by four introns [8].
• Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues [1].

Anatomical context of NIT1

• To probe the capacity of ths enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor [9].

Associations of NIT1 with chemical compounds

• 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 [10].
• Collectively, these data suggest that the elevated levels of IAM in seeds and germinating seedlings result from nitrilase action on indole-3-acetonitrile, a metabolite produced in the plants presumably from glucobrassicin turnover [11].
• The mRNA levels of a diverse array of gene products increases during leaf senescence, including a protease, a ribosomal protein, two cinnamyl alcohol dehydrogenases, a nitrilase and glyoxalase II [12].
• The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA [3].

Other interactions of NIT1

• Genomic Southern hybridization, among other experiments, strongly suggests that tobacco has NIT4 homologs but not NIT1 to NIT3 homologs [7].
• Three (NIT1, NIT2 and NIT3) of the four genes have high similarity [7].
• The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase [10].
• Transcript levels known to be involved in IAA/glucosinolate synthesis and metabolism (nitrilase and myrosinase) were examined and both showed developmental regulation, while only nitrilase mRNA levels differed between wild type and mutant seedlings [13].

Analytical, diagnostic and therapeutic context of NIT1

• Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid [2].

References