Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid.
From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-beta-D-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid.
Bartling
D
D
Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, Bochum, Federal Republic of Germany.
Seedorf
M
M
Mithöfer
A
A
Weiler
E W
EW
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D007210:Indoleacetic Acids / Q000378:metabolism*; D007211:Indoles / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010937:Plant Growth Regulators / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D007210:Indoleacetic Acids; D007211:Indoles; D010937:Plant Growth Regulators; C030737:indoleacetic acid; D004247:DNA; C016516:indole-3-acetonitrile; D000619:Aminohydrolases; C021867:nitrilase