Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg.
The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.
Wohlfarth
G
G
Institut für Mikrobiologie, Universität Stuttgart, Federal Republic of Germany.
Geerligs
G
G
Diekert
G
G
D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010462:Peptostreptococcus / Q000201:enzymology*; D010618:Phenanthrolines / Q000494:pharmacology; D010659:Phenylhydrazines / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013057:Spectrum Analysis; D015032:Zinc / Q000494:pharmacology
D006898:Hydroxylamines; D010618:Phenanthrolines; D010659:Phenylhydrazines; C030299:phenylhydrazine; D019811:Hydroxylamine; D009249:NADP; C002701:neocuproine; D004492:Edetic Acid; D012964:Sodium; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D008628:Mercury; D015032:Zinc