Epimerization of the D-Valine Portion in the Biosynthesis of Actinomycin D

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)

δ‐l‐(α‐Aminoadipoyl)‐l‐cysteinyl‐d‐valine synthetase: isolation of l‐cysteinyl‐d‐valine, a ‘shunt’ product, and implications for the order of peptide bond formation

“…Determinations of incorporation of radiolabel were done in triplicate. N-terminal residue [10]. This is in contrast to e.g.…”

Biosynthesis of Acylpeptidolactones of the Daptomycin Type

“…PM = prestained molecular marker (Sigma); ESF1, 2 = S. frudiae proteins. In both systems analysed here, peptide synthetases have been detected with antibodies derived against the actinomycin synthetase 2 from Sti-eptomyces chrysomallus [30] or a sequence-derived antibody [18]. Both systems contain a large multienzyme activating lactone constituents, and a small multienzyme activating residue 13, L-kynurenine or L-Ile/L-Val, respectively.…”

Microarray Analysis of the Mycobacterium tuberculosis Transcriptional Response to the Acidic Conditions Found in Phagosomes

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