Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.
AUTOR(ES)
Kobayashi, M
RESUMO
Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=45637Documentos Relacionados
- The Nitrilase ZmNIT2 Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid1
- Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.
- Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.
- Effects of site-directed mutagenesis at residues cysteine-31 and cysteine-184 on lecithin-cholesterol acyltransferase activity.
- Uptake of Indole-3-acetic Acid and Indole-3-acetonitrile by Avena Coleoptile Sections 12