Purification and properties of a NADH‐dependent 5,10‐methylenetetrahydrofolate reductase from Peptostreptococcus productus

Abstract

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 pmol . min-' mg protein-' (37°C; pH 5.5). The apparent K , for NADH was near 10 pM. The apparent molecular mass of the enzyme was determined by gel filtration to be ~2 5 0 . 0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD' nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used as substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP+-dependent reductase of eucaryotes investigated so far.

Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups

“…The methylene-H4F reductase has been proposed to be involved in energy generation in acetogens (8). The membrane location of this enzyme in C. thermoautotrophicum and in P. productus is in favor of this suggestion (14,33). Since the extracts of S. sphaeroides were prepared by French pressing, it is not surprising that the majority of the methylene-H4F reductase was recovered from the soluble fraction (data not shown); this is also observed in C. thermoautotrophicum and in P. productus.…”

“…Nothing is known about the physiological electron donor (33). The methylene-H4F reductase has been proposed to be involved in energy generation in acetogens (8).…”

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

“…A. woodii can couple the oxidation of various donors, such as hexoses, methyl group-containing compounds, or H 2 -CO 2 , with caffeate reduction. Oxidation of hexoses generates NADH as well as reduced ferredoxin, and methyl group oxidation yields reduced pyridine nucleotides and probably ferredoxin (47,55). Therefore, oxidation of these substrates can easily be coupled to caffeate reduction directly via NADH.…”

A corrinoid-dependent catabolic pathway for growth of aMethylobacteriumstrain with chloromethane

“…CH 3 -H 4 folate was separated from the incubation mixture by HPLC (14) and detected spectrophotometrically at 320 nm. The incubation mixture contained cell-free extract (0.8-4 mg/ml protein) in 100 mM Tris-SO 4 (pH 7.8) buffer, 5 mM DTT, 2.4 mM H 4 folate [of which 1 mM was biologically available (22)], and 1 mM titanium(III)citrate. Cell-free extracts dialyzed against 100 mM Tris-SO 4 (pH 7.8) were also used to check whether any endogenous cofactors in the extracts were involved in dehalogenase activity.…”