Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis.
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.
Piotrowski
Markus
M
Department of Plant Physiology, Ruhr-Universität, Universitätsstrasse 150, D-44801 Bochum, Germany. Markus.Piotrowski@ruhr-uni-bochum.de
Janowitz
Tim
T
Kneifel
Helmut
H
D017360:Arabidopsis / Q000201:enzymology*; D015317:Biogenic Polyamines / Q000096:biosynthesis*; D015152:Blotting, Northern; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D010802:Phylogeny; D014512:Ureohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015317:Biogenic Polyamines; C027237:N-carbamoylputrescine hydrolase; D014512:Ureohydrolases