THE METHIONINE SALVAGE PATHWAY IN KLEBSIELLA-PNEUMONIAE AND RAT-LIVER - IDENTIFICATION AND CHARACTERIZATION OF 2 NOVEL DIOXYGENASES

The 5-methylthio-D-ribose moiety of 5'-(methylthio)adenosine is converted to methionine in a wide variety of organisms, 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniae and rat liver, This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the alpha-keto acid precursor of methionine), Previously, we had purified an enzyme (E2) from Klebsiella which catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously, This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III, Experiments with O-18(2) show that E2 is a dioxygenase which incorporates one molecule of O-18 into formate and butyric acid, No cofactor has been identified, We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine, The keto acid is probably produced non enzymically in Klebsiella. We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III, This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified, Experiments with O-18(2) show that E3 is a dioxygenase which incorporates an O-18 molecule into formate and the alpha-keto acid, In rat liver CO formation was not detected,