Epimerization of the D-valine portion in the biosynthesis of actinomycin D

Biochemistry. 1994 Aug 9;33(31):9358-64. doi: 10.1021/bi00197a041.

Abstract

In the biosynthesis of actinomycin, the multifunctional actinomycin synthetase II (ACMS II) assembles 4-methyl-3-hydroxyanthranilic acid (4-MHA), L-threonine and D-valine, the first three residues of the 4-MHA peptide lactone chain. ACMS II activates L-threonine and L-valine but not D-valine as thioesters via their adenylates, and there is no epimerization of the covalently bound L-valine. When L-threonine and L-valine are presented to the enzyme together with the 4-MHA analogue p-toluic acid and the 4-MHA-activating enzyme ACMS I, ACMS II forms the two diastereomers p-toluyl-L-Thr-L-Val and p-toluyl-L-Thr-D-Val in equal amounts along with p-toluyl-L-Thr in a cofactor-independent manner. Studies with [2,3-3H2]valine revealed that p-toluyl-L-Thr-D-Val contained approximately 50% of the tritium label found in the LL-diastereomer. Concomitantly, radioactive water was formed due to enzyme-catalyzed hydrogen exchange with the solvent during epimerization. In the absence of threonine (or MgATP), however, the amount of radioactive water formed from [3H]valine was significantly less, which suggests that the peptide bond between L-threonine and L-valine is formed prior to the epimerization at C-2 of valine. The facts that both LL- and LD-acyldipeptides are equally present on the enzyme's surface--as revealed by using 14C-labeled threonine or valine as precursors--and that the L-valine in the LL-diastereomer apparently has not lost hydrogen strongly suggests that the LL-diastereomer is an obligatory intermediate in the formation of the LD-dipeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / chemistry
  • Carbon Radioisotopes
  • Dactinomycin / biosynthesis*
  • Dactinomycin / chemical synthesis
  • Dactinomycin / chemistry
  • Indicators and Reagents
  • Molecular Sequence Data
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Nucleotidyltransferases / isolation & purification
  • Nucleotidyltransferases / metabolism*
  • Oligopeptides / chemistry
  • Peptide Synthases / isolation & purification
  • Peptide Synthases / metabolism*
  • Radioisotope Dilution Technique
  • Stereoisomerism
  • Streptomyces / enzymology*
  • Tritium
  • Valine* / metabolism

Substances

  • Anti-Bacterial Agents
  • Carbon Radioisotopes
  • Indicators and Reagents
  • Multienzyme Complexes
  • Oligopeptides
  • Tritium
  • Dactinomycin
  • Nucleotidyltransferases
  • actinomycin synthetase I
  • Peptide Synthases
  • actinomycin synthetase II
  • Valine