Purification and characterization of 2-keto-d-galactonate reductase from Pseudomonas fluorescens

NADPH-dependent 2-keto-d-galactonate reductase from Pseudomonas fluorescens IFO 14808, which was present in the soluble protein fraction, was purified to apparent homogeneity using a five-step procedure. The enzyme catalyzed NADPH-dependent reduction of 2-keto-aldonate to aldonate as well as NADP+-dependent oxidation of aldonate and aldonate-6-phosphate to 2-keto derivatives. The enzyme consisted of two subunits with molecular masses of about 37 kDa. The gene encoding the enzyme was identified in the complete sequence of the P. fluorescens pf-5 genome based on the N-terminal amino acid sequence derived from the purified enzyme. The enzyme sequence exhibited 75 and 72% amino acid identities to the probable 2-keto-d-gluconate reductase from Pseudomonas aeruginosa and 2-keto-d-gluconate-6-phosphate reductase from Pseudomonas putida, respectively. The enzyme was found to belong to the 2-keto-aldonic acid reductase family in bacteria, although its substrate specificities differed from those of previously characterized members. Its relative reduction activities were 1.0 and 3.9 for 2-keto-d-gluconate and 2-keto-d-galactonate, respectively, while its relative oxidation activities were 1.0, 4.3, 1.4 and 0.4 for d-gluconate, 6-phospho-d-gluconate, d-galactonate and d-galactono-1,4-lactone, respectively. The 6-phospho-d-gluconate reductase activity was inhibited either competitively by 6-phospho-d-glucose and 2-keto-d-gluconate or uncompetitively by dilute 2-keto-d-gluconate. The possibility of d-erythorbic acid formation from d-galactose by P. fluorescens is discussed.