Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).

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1. Two carbons, carbon-2 and one of carbons-3 to -5, of lysine seemed likely to be incorporated into one of carbon of the chromophore moiety of formycin.2. From the results of radioisotopic studies with glutamate, r-amino-n-butyrate or organic acids related to tricarboxylic acid cycle, the important role of glutamate in the biosynthesis of formycin was strongly suggested.3. The incorporation of nitrogen(s) of lysine into three nitrogens, including two nitrogens of pyrazole ring, of formycin was suggested by mass spectroscopy. 4. Ribose was estimated as a direct precursor for the ribosyl moiety of formycin, whereas the biosynthesis of ribose was shown to occur via the pathway other than hexose monophosphate shunt or glucuronate pathway.5. In replacement culture, the salvage synthesis of formycin from its chromophore moiety was not observed and it was also evident that the chromophore moiety or pyrazofurin (pyrazomycin) inhibited the biosynthesis of formycin.There have been several investigations on the biosynthesis of C-riboside nucleosides, i.e., pseudouridine, showdomycin, minimycin, pyrazofurin (pyrazomycin) and the formycin family. In the biosynthesis of pseudouridine, the intramolecular rearrangement in tRNA of uridylate to pseudouridylate has been strongly suggested.1-6) The biosynthesis of showdomycin has been well established and confirmed to occur by the condensation of ribose and an asymmetric four-carbon dicaboxylic acid.7-10)Recently, studies on the biosynthesis of minimycin revealed a novel mechanism whereby C-riboside linkage is formed from sedoheptulose 7-phosphate (or eight-carbon branched chain sugar phosphate).11) C-Riboside linkage of pyrazofurin was also believed to be formed by this mechanism.11)The biosynthesis of formycin by Nocardia interforma has been proposed to occur via a pathway other than that of biosynthesis of purine nucleotide while exogeneously added ribose was estimated to be incorporated into the ribosyl moiety of formycin.12) It was also reported that formycin may be formed from formycin 5'-monophosphate then deaminated to formycin B.13) Using Streptomyces sp.MA406-A-1, a formycin-producing strain, we reported that carbon atom from lysine, glutamate and/or aspartate was incorporated into formycin with high efficiency, and that novel enzyme(s) catalyzing the amination of formycin B to formycin was present in this organism. 14,15) To elucidate the more detailed mechanism of formycin biosynthesis, we examined the incorporation and the distribution of various labeled compounds into formycin and this paper presents the results of these studies.

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