Biosynthesis of quinoxaline antibiotics: purification and characterization of the quinoxaline-2-carboxylic acid activating enzyme from Streptomyces triostinicus

Abstract

A quinoxaline-2-carboxylic acid activating enzyme was purified to homogeneity from triostin-producing Streptomyces triostinicus. It could also be purified from quinomycin-producing Streptomyces echinatus. Triostins and quinomycins are peptide lactones that contain quinoxaline-2-carboxylic acid as chromophoric moiety. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on quinoxaline-2-carboxylic acid and the formation of the corresponding adenylate. Besides quinoxaline-2-carboxylic acid, the enzyme also catalyzes the formation of adenylates from quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid. No adenylates were seen from quinoline-3-carboxylic acid, quinoline-4-carboxylic acid, pyridine-2-carboxylic acid, and 2-pyrazinecarboxylic acid. Previous work [Gauvreau, D., & Waring, M. J. (1984) Can. J. Microbiol. 30, 439-450] revealed that quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid became efficiently incorporated into the corresponding quinoxaline antibiotic analogues in vivo. Together with the data described here, this suggests that the enzyme is part of the quinoxaline antibiotics synthesizing enzyme system. The enzyme displays a native molecular weight of 42,000, whereas in its denatured form it is a polypeptide of Mr 52,000-53,000. It resembles in its behavior actinomycin synthetase I, the chromophore activating enzyme involved in actinomycin biosynthesis [Keller, U., Kleinkauf, H., & Zocher, R. (1984) Biochemistry 23, 1479-1484].

A Nonribosomal System of Peptide Biosynthesis

“…These transferases employ acyl-CoA, while several aromatic acyl groups of peptides seem to be introduced as enzymebound adenylates [200]. Such systems, including enterobactin (EntE) [ 1721, actinomycin (ACMSI) 12011, triostin (TRSI) [212], mikamycin (MKMS) [203], nosiheptide and thiostreptone 1224, 2251 are involved in the initiation of cyclic chromopeptides by acylation of the first amino acid, thioester bound to the respective peptide synthetase. This reaction has been studied by Keller and Stindl and requires the presence of the cofactor phosphopantetheine [ 2001.…”

Functional Cross-talk between Fatty Acid Synthesis and Nonribosomal Peptide Synthesis in Quinoxaline Antibiotic-producing Streptomycetes

“…Streptomyces griseoviridus DSM40229 was from the Deutsche Sammlung fü r Mikroorganismen (Braunschweig, Germany). Maintenance, preparation of inocula for liquid culture of S. echinatus, and S. triostinicus, as well as growth in liquid cultures were carried out essentially as described previously, except that S. echinatus liquid cultures were incubated for up to 38 h depending on the maximum rate of [methyl- 14 C]methionine incorporation into echinomycin during short term incorporation experiments (14). Streptomyces lividans TK64 and Streptomyces coelicolor M145 were obtained from the John Innes strain collection (Norwich, United Kingdom).…”

“…Radioactive determinations of filters or of liquid samples were achieved by liquid scintillation counting using a Wallac scintillation system. Echinomycin production in cultures was monitored by short term incorporation tests as described (14). Echinomycin or triostin extracted from cultures was identified by TLC along with authentic standards using solvent system I. Amino acids were chromatographed on silica gel plates using solvent system II.…”

“…The ATP-pyrophosphate exchange reaction dependent on the aromatic carboxylic acids was carried out as described previously (14,20). For routine measurements of enzyme thioester formation, the standard filter binding assay was used as described previously (20).…”

“…Triostin synthetase I (TrsI), a 58-kDa protein present in triostin A-producing S. triostinicus and in echinomycin-producing S. echinatus, activates QA as the adenylate (14). It can also activate a number of structural analogues of QA such as quinoline-2-carboxylic acid, thieno [3,2b]pyridine-5-carboxylic acid (TPA), and 3-hydroxyquinoline-2-carboxylic acid.…”