Epimerization of the D-valine portion in the biosynthesis of actinomycin D.
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)
Stindl
A
A
Institut für Biochemie, Technische Universität Berlin, Germany.
Keller
U
U
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D002250:Carbon Radioisotopes; D003609:Dactinomycin / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D010453:Peptide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology*; D014316:Tritium; D014633:Valine / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D009842:Oligopeptides; D014316:Tritium; D003609:Dactinomycin; D009713:Nucleotidyltransferases; C075125:actinomycin synthetase I; D010453:Peptide Synthases; C052258:actinomycin synthetase II; D014633:Valine