Isolation and characterization of tryptophan transaminase and indolepyruvate C methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus

2 Enzymes, tryptophan transaminase and indolepyruvate C methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell free extracts of S. griseus. The transaminase has been purified 3 fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the α ketoglutarate and pyridoxal phosphate dependent transamination of L-tryptophan, 3 methyltryptophan, L-phenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110 fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromatography, and Bio Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 ± 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,500 ± 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5° but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p chloromercuribenzoate and N-ethylmaleimide. The Zn²⁺ and Fe²⁺ chelators 1,10 phenanthroline and 2,2' bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis Menten constants have been determined for the 110 fold purified enzyme as 1.2 x 10^-6 M for S-adenosylmethionine and 4.8 x 10^-6 M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki = 2.3 mM) and L-tryptophan (Ki = 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephadex G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p hydroxyphenylpyruvate but this capability is lost upon purification of the indolepyruvate C-methyltransferase activity. No methylation of L-tryptophan occurs under the conditions used.