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B6db references: 76025004

type Journal Article
authors Speedie, M. K.; Hornemann, U.; Floss, H. G.
title Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus
journal J Biol Chem
ui 76025004
year (1975)
volume 250
number 19
pages 7819-25.
keywords Antibiotics/biosynthesis
abstract 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.
last changed 2002/11/13 15:40

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