Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition

Abstract

The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with ['85W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of '85W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Ful was catalyzed by two different dehydrogenases.Pseudomonas putida Ful was isolated from an enrichment culture with 2-furoic acid as the sole source of carbon and energy (24). The formation of 2-furoyl-coenzyme A (CoA) is the first reaction step in 2-furoic acid degradation. The second enzymatic step is catalyzed by a dehydrogenase that introduces a hydroxyl group to form 5-hydroxy-2-furoylCoA (23,24,49,50). Other substrates for P. putida Ful were furfuryl alcohol and furfural, which were degraded via 2-furoic acid and 2-furoyl-CoA (Fig.

Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14

“…Arsenite (1 mM) was added to inhibit 2-oxoglutarate dehydrogenase. After overnight incubation 3 mM of 2-oxoglutarate had accumulated, which is in agreement with previous experiments performed on P. putida Fu1 (14). No 2-oxoglutarate was formed with control cells of P. putida S12 expressing HmfABCD.…”

“…Mutant phenotypes of transposon mutants and BLASTx analysis (17) of the genes included in the two clusters are summarized in Table 1. The putative enzyme functions encoded by the hmfABCDE cluster of C. basilensis HMF14 were in good agreement with the enzyme activities that were reported to constitute the furoicacid degradation pathway of Pseudomonas putida strains F2 and Fu1 (14,15) (Fig. 2B).…”

“…Cell extracts of both wild-type C. basilensis HMF14 and P. putida S12 expressing HmfFG formed 2-furoic acid when incubated with FDCA (6.0 AE 0.1 U · g −1 and 8.6 AE 0.7 U · g −1 , respectively). HMF acid was not decarboxylated to furfuryl alcohol, demonstrating that (14). Colored hexamers and triangles indicate enzymes with the following activities: orange hexagon, furfural/HMF oxidoreductase; red and green triangles, 2,5-furan-dicarboxylic acid decarboxylase; blue triangle, 2-furoyl-CoA synthetase; yellow triangle, furoyl-CoA dehydrogenase; purple triangle, 2-oxoglutaryl-CoA hydrolase.…”

“…A number of microorganisms that metabolize furfural or HMF have been described (9,10,13,14). A furfural degradation pathway has been proposed based on enzyme activities in Pseudomonas putida strains Fu1 (14) and F2 (15). No reports are available on enzymes involved in HMF degradation, and no pathway for the degradation of HMF has been proposed to date.…”

“…Bioabatement with microorganisms that degrade furan derivatives may present an important alternative approach (8)(9)(10)(11)(12). A number of microorganisms that metabolize furfural or HMF have been described (9,10,13,14). A furfural degradation pathway has been proposed based on enzyme activities in Pseudomonas putida strains Fu1 (14) and F2 (15).…”