Formate Dehydrogenase from Pseudomonas oxalaticus

Abstract

Formate dehydrogenase (EC 1.2.1.2) from Pseudornonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 31 5 000) is a complex flavoprotein containing 2 FMN, 18-25 non-heme iron atoms and 15-20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD', oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors.The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species.When the aerobic bacterium Pseudornonas osalati-L'US is grown on formate as the main carbon source, the oxidation of formate to COZ is the key process for obtaining energy and reducing equivalents. The key enzyme for the latter is formate dehydrogenase, a complex flavoprotein containing FMN, iron and labile sulphur. It catalyzes the reaction HCOF + NAD' COz + NADH. (1) The C02 formed is incorporated predominantly into the ribulose 1,5-bisphosphate cycle as in autotrophic organisms [I, 21. The observation that a cosubstrate (pyruvate or acetate) is needed for growth on formate [3] has not been related to the carbon metabolism of the organism so far.In an earlier paper [4] we reported that the attainment of the thermodynamically expected equilibrium (1) is catalyzed by the enzyme in both directions and that COZ is reduced to formate by NADH under catalysis of the enzyme. COZ (not bicarbonate) has been identified as the active species in this equilibrium.As observed for all other complex flavoproteins, formate dehydrogenase catalyzes several other nonphysiological oxidation/reduction reactions because its chromophoric groups interact with various electron donors and acceptors : dyes, oxygen, ferricyanide. The resulting reactions can be separated into two groups,

Purification and Characterization of a NAD+-Dependent Formate Dehydrogenase Produced byParacoccussp.

“…The molecular weight of the purified enzyme was 100,000 by Sephadex G-150 gel filtration and 49,000 by SDS-PAGE, therefore, the FDH probably has two identical subunits like FDHs from Candida boidinii, 8) Achromobacter parvulus, 4) and Moraxella Sp.,13) but different from Pseudomonas oxalaticus. 12 ) Although the specific activity of the purified enzyme (11.6 U/mg at 30°C) is not as high as that of Ps. oxalaticus (42 U/mg at 25°C) of a formate-assimilating bacterium, it is superior to those of FDHs from methanol-utilizing microbes of C. boidinii 8 ) (2.4 U/mg at30°C), A. parvulus (11.0 U/mg at 37°C), and Moraxella sp.…”

“…Therefore, we tried to isolate microorganisms with enzymes of higher specific activity. Many methylotrophs such as Achromobacter parvulus, 4,5) Methylobacterium organophilum,6) Kloeckera Sp.,7) C. boidinii, 8,9) and Pichia pastoris,10) and methanogenic bacteria such as Methanobacterium formicicum11) have been isolated to get FDH, but few bacteria that assimilate formate and produce FDH have been isolated (Pseudomonas oxalaticus 12 ) and Moraxella Sp.13)). We considered that formate-assimilating bacteria have not only higher enzyme productivity than plants but also probably stronger FDH activity than methanol-utilizing or methanogenic bacteria, because they oxidize formate directly.…”

Formate production through biocatalysis

“…Formate dehydrogenases are enzymes with the potential to catalyze this reaction; however they generally favor the reverse process, i.e., formate oxidation. By contrast, the formate dehydrogenase of the acetogen Clostridium carboxidivorans has been found to preferentially catalyze the reduction of CO 2 . This is in accord with its natural role to introduce CO 2 as a carbon source in the Wood-Ljungdahl pathway.…”

“…The direction of catalysis derives from the enzyme's low affinity for formate. This enzyme is therefore an excellent candidate for biotechnological applications aimed at producing formic acid and derivative chemicals from CO 2 .…”

Isolation, Purification, and Partial Characterization of Formate Dehydrogenase from Soybean Seed

“…The enzyme occurs in bacteria (6,11), yeast (16,21), plants (1,4,12,18), and animals (9). It has been isolated and partially purified from mung beans (14), yeast (16), peas (9,19) and Pseudomonas oxalaticus (1 1).…”