Characterization and synthetic applications of recombinant AtNIT1 from <i>Arabidopsis thaliana</i>

Oßwald, Steffen; Wajant, Harald; Effenberger, Franz

doi:10.1046/j.0014-2956.2001.02702.x

Cited by 80 publications

(52 citation statements)

References 43 publications

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“…Indeed, it was shown conclusively by GC-MS that, besides IAA, IAM is a true reaction product of all three nitrilase isoenzymes, when IAN is used as substrate. This observation was independently verified for NIT1 in a recent study appearing in print during the preparation of this manuscript (Osswald et al 2002). IAM went undetected by the GC-MS technique used in our previous work which included methylation of samples and focussed on acidic metabolites (Vorwerk et al 2001).…”

Section: Discussionsupporting

confidence: 61%

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

Pollmann

Müller

Piotrowski

et al. 2002

Planta

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An HPLC/GC-MS/MS technique (high-pressure liquid chromatography in combination with gas chromatography-tandem mass spectrometry) has been worked out to analyze indole-3-acetamide (IAM) with very high sensitivity, using isotopically labelled IAM as an internal standard. Using this technique, the occurrence of IAM in sterile-grown Arabidopsis thaliana (L.) Heynh. was demonstrated unequivocally. In comparison, plants grown under non-sterile conditions in soil in a greenhouse showed approximately 50% higher average levéis of IAM, but the differences were not statistically significant. Thus, microbial contributions to the IAM extracted from the tissue are likely to be minor. Levéis of IAM in sterile-grown seedlings were highest in imbibed seeds and then sharply declined during the first 24 h of germination and further during early seedling development to remain below 20-30 pmol g l fresh weight throughout the rosette stage. The decline in indole-3-aetic acid (IAA) levéis during germination was paralleled by a similar decline in IAM levéis. Recombinant nitrilase isoforms 1, 2 and 3, known to synthesize IAA from indole-3-acetonitrile, were shown to produce significant amounts of IAM in vitro as a second end product of the reaction besides IAA. NIT2 was earlier shown to be highly expressed in developing and in matare A. thaliana embryos, and NIT3 is the dominantly active gene in the hypocotyl and the cotyledons of young, germinating seedlings. Collectively, these data suggest that the elevated levéis 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.

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Section: Discussionsupporting

confidence: 61%

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

Pollmann

Müller

Piotrowski

et al. 2002

Planta

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“…In contrast, 2-fluoroarylacetonitriles were preferentially converted to the amides and only minor amounts of the corresponding carboxylic acids were formed. It was suggested that the increased formation of the amides was due to the presence of highly electron-withdrawing substituents such as fluorine or nitro-groups (Effenberger & Osswald, 2001;Osswald et al, 2002). The results of the present study substantiate this observation and demonstrate that it may be possible to utilize classical nitrilases with certain substrates as nitrile hydratases forming stoichiometric amounts of the corresponding amides.…”

supporting

confidence: 78%

“…Furthermore, a ricinine-specific nitrilase from Pseudomonas sp. released the corresponding amide (7-10 % of the amount of the acid formed) and two nitrilases from the plant Arabidopsis thaliana hydrolysed 2-fluoroacetonitriles (AtNit1) or bcyano(L-)-alanine (AtNit4) to significant amounts of the corresponding amides Effenberger & Osswald, 2001;Osswald et al, 2002;Piotrowski et al, 2001). The nitrilase from strain EBC191 demonstrated a clear correlation between the structure of the substrates and the relative proportion of the respective amides formed.…”

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confidence: 99%

Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme

et al. 2005

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The gene encoding an enantioselective arylacetonitrilase was identified on a 3?8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts of L-rhamnose-induced cells of Escherichia coli JM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 6C), pH optimum (pH 6?5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme from P. fluorescens EBC191. The enzyme hydrolysed various phenylacetonitriles with different substituents in the 2-position and also heterocyclic and bicyclic arylacetonitriles to the corresponding carboxylic acids. The conversion of most arylacetonitriles was accompanied by the formation of different amounts of amides as by-products. The relative amounts of amides formed from different nitriles increased with an increasing negative inductive effect of the substituent in the 2-position. The acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus mandelonitrile was converted by the nitrilase preferentially to R-mandelic acid and S-mandelic acid amide. The nitrilase gene is physically linked in the genome of P. fluorescens with genes encoding the degradative pathway for mandelic acid. This might suggest a natural function of the nitrilase in the degradation of mandelonitrile or similar naturally occurring hydroxynitriles.

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“…Phenylacetonitrile 1 was a rather poor substrate for NIT2 compared to the other substrates in this series. In accordance to literature reports, [30,15] 4-phenylbutyronitrile 2 was converted approximately five times faster than 1 with the wild-type enzyme. The activity for 2 has been improved six times using the mutant 7/I2B.…”

supporting

confidence: 76%