The methionine salvage pathway in Klebsiella pneumoniae and rat liver. Identification and characterization of two 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 18O2 show that E2 is a dioxygenase which incorporates one molecule of 18O 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 18O2 show that E3 is a dioxygenase which incorporates an 18O molecule into formate and the alpha-keto acid. In rat liver CO formation was not detected.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
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JW
Wray
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Abeles
D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D049308:Dioxygenases; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D008715:Methionine; D010105:Oxygenases; D049308:Dioxygenases; C083734:aci-reductone oxidase (CO-forming)