Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway.
Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
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D002247:Carbon Isotopes; D005561:Formates / Q000378:metabolism; D006859:Hydrogen; D007553:Isotope Labeling / Q000379:methods; D007711:Klebsiella pneumoniae / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008715:Methionine / Q000096:biosynthesis*; D010103:Oxygen Isotopes; D010105:Oxygenases / Q000378:metabolism; D013865:Thioglycosides / Q000378:metabolism*
D002247:Carbon Isotopes; D005561:Formates; D010103:Oxygen Isotopes; D013865:Thioglycosides; C030544:formic acid; C001955:5-methylthioribose; D006859:Hydrogen; D008715:Methionine; D010105:Oxygenases