Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT 2/ NIT 1/ NIT 3-gene cluster

Abstract

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5'-->3' direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 degrees C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 degrees C) and PPN (35-40 degrees C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of beta-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.

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

“…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).…”

“…In a preliminary earlier report, it was stated that all four A. thaliana nitrilases would convert IAN to IAA with a side-product of IAM noted on thin-layer (Bartel and Fink 1994). However, this reaction product was neither identified unambiguously in that study ñor has it been confirmed in later studies that NIT4 uses IAN as a substrate (Vorwerk et al 2001). Thus, a re-examination was carried out using recombinant NIT1, NIT2 and NIT3 in highly purified form.…”

“…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).…”

“…Cloning of the A. thaliana nitrilase isoforms into the pET21-b( + ) vector (Novagen, Madison, Wis., USA) was as described earlier (Vorwerk et al 2001). For the expression of nitrilase enzymes, the E. coli strain BL21-CodonPlus (DE3)-RIL (Stratagene, La Jolla, Calif., USA) was used.…”

“…Thus, a re-examination was carried out using recombinant NIT1, NIT2 and NIT3 in highly purified form. To ease purification, all nitrilases were expressed from their cDNAs as C-terminally hexahistidine-tagged fusión proteins (Vorwerk et al 2001) and were purified to apparent homogeneity as judged by SDS-PAGE (data not shown). Enzyme assays with the purified nitrilases gave proof of the formation of both IAM and IAA as end products of the reactions of each of the three nitrilase isoforms (Fig.…”