Reduction of aromatic acids to aldehydes and alcohols in the cell-free system. 1. Purification and properties of aryl-aldehyde: NADP-oxidoreductase from Neurospora crassa

Abstract

An enzyme catalyzing the reduction of the carboxyl group of aromatic acids to the respective aldehydes has been isolated from mycelia of the fungus Neurospora crassa grown in the presence of salicylic acid as an inducer. This protein has been purified about 300-fold by precipitation of nucleic acids with protamine sulfate, ammonium sulphate fractionation, adsorption on calcium-phosphate gel and chromatography on TEAE-cellulose (at pH 7.6 and 8.4) and on hydroxylapatite. The enzyme, the molecular weight of which was estimated to be about 120,000 by chromatography on a calibrated Sephadex G-200 column requires NADPH and ATP as cofactors. The Km-values for salicylic acid as a substrate are 1.67 × 10−4 M and for ATP 1.52 × 10−4 M. The presence of Mg-ions and of a reduced thiol in the incubation mixture is necessary to achieve full activity. The purified enzyme is most active around pH 8.0 and 35°. It shows a broad range of substrate specificity towards aromatic acids, especially to phenyl carboxylic and phenyl acrylic acids, but does not catalyze the reduction of aliphatic carboxyl groups. During the reaction ATP is split into ADP and inorganic phosphate, while stoichiometric amounts of aldehyde are produced from the corresponding acid. NADPH used as a reductant, the formation of the carbonyl group involves the oxidation of the hydrogen donor in a molar ratio of 1:1. Since no intermediate could be detected and there was no evidence for a reverse reaction we formulate the reaction as R–COOH + ATP + NADPH + H+→ R-CHO + ADP + NADP++ PO43-+ H2O; the systematic name aryl aldehyde: NADP oxidoreductase (ADP) is proposed; the enzyme belongs to EC group 1.2.1. By supplying α-, or β-[3H]NADP the enzyme was shown to be of the “B-type”.