Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus.
Two 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 Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, 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 chromotography, 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 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees 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(-5) 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 equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades 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.
D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002614:Chelating Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007211:Indoles / Q000096:biosynthesis; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011773:Pyruvates; D013056:Spectrophotometry, Ultraviolet; D013305:Streptomyces griseus / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism; D014364:Tryptophan
D000900:Anti-Bacterial Agents; D002614:Chelating Agents; D007211:Indoles; D011773:Pyruvates; D013439:Sulfhydryl Reagents; D014364:Tryptophan; D008780:Methyltransferases; D000637:Transaminases; D004121:Dimethyl Sulfoxide; C001447:indolmycin