Antimycobacterial activity of lipodepsipeptides produced by Pseudomonas syringae pv syringae B359.

Some lipodepsipeptides produced by Pseudomonas syringae pv. syringae showed strong antimycobacterial activity towards Mycobacterium smegmatis. MIC values found were between 1.5-3.2 microg/ml, which is comparable to some primary drugs for tuberculosis. Among the lipodepsipeptides, Syringomycin E (SRE) appears to be the most potent antimycobacterial agent.

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)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for M(r) estimations of high-molecular-weight polypeptides.

A convenient method was established for the M(r) estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of large polypeptides in the range from 250,000 to 600,000. Such polypeptides had previously been shown to migrate improperly in various gel electrophoresis systems. In a conventional Laemmli system with 3% polyacrylamide, approximately linear log M(r) vs Rf plots could be obtained by varying the gel thickness which ranged from 0.4 to 1.5 mm. In the additional presence of an electrolyte, such as NaCl, running times were shorter and for evaluation linear plots could be constructed from runs in very thin gels with less than 0.5 mm thickness. Handling of gels was greatly facilitated by fixing them to one of the gel chamber glass plates via bind-silane treatment prior to casting.

The initiation of peptide formation in the biosynthesis of actinomycin.

Actinomycin Synthetase II (ACMS II), which activates threonine and valine by a thioltemplate mechanism during the synthesis of the actinomycin half-molecule 4-methyl-3-hydroxyanthranilic acid (4-MHA) pentapeptide lactone, was purified to near homogeneity from Streptomyces chrysomallus. It is a single polypeptide chain of M(r) 280,000 and contains 4'-phosphopantetheine as a covalently bound prosthetic group. ACMS II charges itself with threonine but not with the 4-MHA analogue p-toluic acid via a specific sulfhydryl group at the expense of ATP. Charging of ACMS II with p-toluic acid in thioester linkage took place, however, only when actinomycin synthetase I (ACMS I), a 4-MHA-AMP ligase, was present. In the additional presence of L-threonine, enzyme-bound p-toluyl-L-threonine was formed on ACMS II. The latter compound was also formed when chemically synthesized p-toluic acid adenylate was added instead of ACMS I and p-toluic acid. This indicates that p-toluic acid adenylate is a free intermediate in the reaction and that charging of the enzyme and acylation of threonine are both catalyzed by ACMS II rather than by ACMS I. Chemically synthesized thioesters of p-toluic acid and coenzyme A, pantetheine, or beta-alanyl-cysteamine reacted with ACMS II, threonine, and ATP with formation of enzyme-bound p-toluyl-threonine. In contrast, p-toluyl-cysteamine thioester was inactive, which indicates structural constraints in the reactivity of free thioesters of p-toluic acid with ACMS II. Such constraints obviously require structural similarity of the artificial substrate to a p-toluic acid thioester formed on the enzyme's surface in the course of the reaction. Since free coenzyme A was not involved in the charging of p-toluic acid or in p-toluyl-threonine formation, the sulfhydryl group of the 4'-phosphopantetheine cofactor is most likely the primary acceptor of p-toluic acid (or 4-MHA) in the initiation of peptide lactone formation.