Glutathione-independent isomerization of maleylpyruvate by Bacillus megaterium and other gram-positive bacteria

Abstract

Maleylpyruvate, the ring fission product of gentisic acid, was found to be isomerized to fumarylpyruvate without a requirement for glutathione by an enzyme activity found in cell extracts of m-hydroxybenzoate-grown Bacillus megaterium 410. The isomerization reaction was detected as a shift in the absorbance maximum from 330 nm, the maximum for maleylpyruvate, to 345 nm, the maximum for fumarylpyruvate, when assayed at pH 8.0. Ammonium sulfate precipitation and dialysis of B. megaterium cell extracts resolved the isomerase activity from low-molecular-weight compounds such as glutathione but did not eliminate the isomerase activity. Iodoacetate and p-chloromercuribenzoate were potent inhibitors of the isomerase from B. megaterium. However, N-ethylmaleimide and iodoacetamide did not significantly inhibit this activity. In addition, fumaric acid was demonstrated as a product of gentisate oxidation by dialyzed cell extracts of B. megaterium. Glutathione-independent maleylpyruvate isomerases with properties similar to the isomerase found in B. megaterium were also found in other genera of gram-positive organisms. Eleven different organisms representing the genera Bacillus, Arthrobacter, Corynebacterium, Nocardia, and Rhodococcus were all found to possess this novel type of glutathione-independent maleylpyruvate isomerase.

Glutathione-independent maleylacetoacetate isomerase in gram-positive bacteria

“…In another paper (7) we provide evidence that gram-positive bacteria isomerized maleylpyruvate to fumarylpyruvate without using glutathione as a cofactor. The approaches used included the demonstration of fumarylpyruvate as an intermediate in gentisate oxidation and the identification of fumaric acid as a product.…”

“…Organisms were maintained and grown as previously described (7), by using a minimal medium containing 0.05% L-tyrosine supplemented with 0.005% yeast extract. Cell extracts were prepared as previously described (7), by the method of Hughes (8), and protein concentrations were determined by the method of Lowry et al (10).…”

“…OA3 as described previously for the isolation of maleylpyruvate from gentisate (7). Solutions of fumarylacetoacetate were prepared daily with 0.10 mM maleylacetoacetate in 50 mM Tris buffer (pH 8) and 0.17 mg of protein per ml of ammonium sulfate-precipitated (20 to 60o saturation) and dialyzed cell extracts of L-tyrosine-grown Nocardia globerula CL1.…”

“…Maleylpyruvate isomerase activity was assayed as described elsewhere (7). Products of homogentisate oxidation were recovered by methods previously described (7) and were identified by descending paper chromatography with eucalyptol-n-propanol-formic acid-water (10:10:4:1).…”

“…Maleylpyruvate isomerase activity was assayed as described elsewhere (7). Products of homogentisate oxidation were recovered by methods previously described (7) and were identified by descending paper chromatography with eucalyptol-n-propanol-formic acid-water (10:10:4:1). Fumaric acid produced from homogentisate was detected as a UV absorbing spot which produced a yellow spot against a blue background when sprayed with a solution of 0.05% bromocresol green in ethanol (fumaric acid Rf, 0.81; maleic acid Rf, 0.50).…”