Isolation and characterization of indole-3-acetaldehyde reductases from Cucumis sativus.
In a continuing study of the biosynthetic pathway and regulatory mechanisms governing indole-3-acetic acid (auxin) formation, we report the isolation and initial characterization of three distinct indole-3-acetaldehyde reductases from cucumber seedlings. These enzymes catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H to NAD(P)+. Two of the reductases are specific for NADPH as second substrate, while the third is specific for NADH. The enzymes show a strong specificity for indoleacetaldehyde, with apparent Km values of 73mum, 130mum, and 400mum being calculated for the two NADPH-specific reductases and the NADH-specific reductase, respectively. Under no conditions of substrate concentration, incubation time, or assay method could the reverse reaction be observed. Chromatography on a calibrated Sephadex gel column led to estimated molecualr weights of 52,000 and 17,000 for the NADPH-specific reductases, while a value of 33,000 was obtained for the NADH-specific reductase. Both NADPH-specific reductases showed a pH optimum of 5.2 with a secondary optimum at 7.0, and both enzymes were activated by increasing ionic strength. The NADH-specific reductase showed a pH optimum of 7.0 with a secondary optimum at 6.1 and was slightly inhibited by increasing ionic strength.
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D010944:Plants / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D012965:Sodium Chloride; D000445:Aldehyde Oxidoreductases; C023948:indole-3-acetaldehyde reductase