Abstract
A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.
Highlights
The term “C-N Hydrolases” was introduced by Bork and Koonin in 1994 [1] describing a protein superfamily whose members are all involved in the cleavage of C-N bonds but display only moderate homology (12–24% amino acid identity)
Of the three nitrilase-like proteins (NLPs) existing in A. thaliana (NLP1, gene At2g27450; NLP2, gene At4g08790; and NLP3, gene At5g12040), NLP1 is annotated as a putative nitrilase and shows high homology to an EST clone from tomato, which is annotated as -alanine synthase (88% identity, GenBankTM accession number Y19104)
These enzymes have in common the fact that they hydrolyze N-carbamoyl compounds, and it could be speculated that the natural substrate for NLP1 and its tomato homolog is an N-carbamoyl compound
Summary
The term “C-N Hydrolases” was introduced by Bork and Koonin in 1994 [1] describing a protein superfamily whose members are all involved in the cleavage of C-N bonds but display only moderate homology (12–24% amino acid identity). The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. Like the homologous -alanine synthases, NLP1 showed positive cooperativity toward its substrate.
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