Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are l ocated on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5 '-->3' direction) encode highly similar polypeptides. Copy DNAs encompassin g the entire coding sequences for all three nitrilases were expressed in Es cherichia coli as fusion proteins containing a C-terminal hexahistidine ext ension. All three nitrilases were obtained as enzymatically active proteins , and their characteristics were determined, including a detailed comparati ve 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), allylcy anide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affi nity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown. or they are c lose relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is a ssigned to be the conversion to carboxylic acids of nitriles from glucosino late turnover or degradation. While all nitrilases exhibit a similar pH opt imum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimu m around 30 degreesC independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 degreesC) an d PPN (3540 degreesC). 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 develo ping embryos and in germinating seeds, as judged from an analysis of beta - glucuronidase reporter gene expression under the control of the promoters o f the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification.