Lipoamide dehydrogenase of the phytopathogenic fungi Pythium ultimum and Phytophthora erythroseptica: Differences in conformation and kinetics

Abstract

1. 1. Lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) was isolated from two closely related phytopathogenic fungi, Pythium ultimum and Phytophthora erythroseptica. The enzymes of both microorganisms differ considerably in their catalytic properties, the lipoamide dehydrogenase of P. ultimum being much more active (catalytic centre activity estimated for the reduction of lipoate by NADH: 5900 min −1; for the reduction of NAD + by reduced lipoamide: 100 000 min −) than that of Ph. erythroseptica (corresponding values, 720 min −1 and 4200 min −1). The Michaelis constants for both reactions were determined. 2. 2. Under identical conditions the fluorescence intensity of the enzyme from Ph. erythroseptica at 20°C is about three times higher than that of lipoamide dehydrogenase from P. ultimum. Upon anaerobic reduction with NADH, the Phytophthora enzymes shows beyond 500 nm the typical spectrum of the two equivalent reduced enzyme as reported for the pig heart enzyme (cf. Massey, V., Gibson, Q. H. and Veeger, C. (1960) Biochem. J. 77, 341–351 (ref. 1)), the Pyhthium enzyme becoming more bleached in the flavin region without being fully reduced. 3. 3. At pH 7.6 in phosphate buffer, higher concentrations of NAD + inhibit the activity of the lipoamide dehydrogenase from both fungi when lipoate is reduced with NADH. At pH 5.9 (optimum), NAD + stimulates the reaction in the case of the P. ultimum enzyme, but inhibits the Ph. erythroseptica enzyme. The results indicate that both enzymes have two NAD + binding sites. 4. 4. The results underline the strikingly high genetic variability within the fungus subfamily Pythieae.