Phosphinothricin-tripeptide (PTT) Biosynthesis

A model for the evolution of secondary metabolite biosynthesis

Streptomyces viridochromogenes produces the peptide antibiotic Phosphinothricin-tripeptide (PTT), which is composed of the unusual amino acid phosphinothricin (PT) and two alanine residues. PT is homologous to glutamic acid and thus competitively inhibits bacterial and plant glutamine synthetases (Fig. 1).

Fig. 1: Chemical structures of PTT, PT and glutamic acid.

Because of the herbicidal activity, the antibiotic finds its application as herbicide (BASTA^® (PT) and Herbiace^® (PTT)). The biosynthesis starts with the synthesis of the unusual amino acid N-acetyl-demethyl-phosphiniothricin (N-Ac-DMPT), which is then linked to two alanine residues via non-ribosomal peptide synthetases (NRPS). Within the PTT biosynthesis, two characteristics are found, which enable the analysis of the evolution of secondary metabolism biosynthesis: three enzymes are involved in the biosynthesis of N-Ac-DMPT, which are directly derived from enzymes of the primary metabolic tricarbonic acid cycle (TCA) cycle and which catalyze similar reactions (Fig. 2). Especially the role of an aconitase is a central point of our studies regarding the changes in substrate specificity during evolution and the differentiation-dependent regulation of the enzymes.

Fig. 2: Comparison of biosynthetic Step 6, 7 and 8 of PTT biosynthesis to corresponding reactions of the TCA cycle.

In addition, the PTT biosynthesis represents the only known system, where all peptide synthetase modules, as well as the thioesterase-function are located on separate proteins. In this case, the ‘single enzyme system’ might be an archetype of the non-ribosomal peptide synthesis. Therefore, we are studying the interaction of the single enzymes with the aim to reproduce the evolution of this ‘single enzyme system’ to the multimodular peptide synthetases of NRPS complexes.

Regulatory function of S. viridochromogenes aconitase AcnA

In many organisms, aconitases have dual functions; they serve as enzymes in the tricarboxylic acid cycle and as regulators of iron metabolism. Currently, we are investigating the role of the aconitase AcnA in S. viridochromogenes. AcnA belongs to the iron regulatory proteins (IRPs). In addition to its catalytic function, AcnA plays a regulatory role by binding to iron responsive elements (IREs) located on the untranslated region of certain mRNAs (Fig. 3). With the bioinformatical tool SPIRE we are able to identify IRE-like structures in the S. viridochromogenes genome as well as in other Streptomyces genomes. One of the identified IRE-structures is located upstream of recA, which is involved in the bacterial SOS response, and another one is upstream of ftsZ, required for the onset of sporulation in streptomycetes. We already proved the functionality of different IRE structures with gel shift assays and identified specific IRE consensus sequences. Furthermore, we showed that RecA is up-regulated on posttranscriptional level under oxidative stress conditions in the wild-type strain but not in the acnA mutant, suggesting a regulatory role of AcnA in oxidative stress response.

Fig. 3: Schematic presentation of the conserved protein regions in AcnA (front view). AcnA was modeled according to the known structure of rabbit IRP1. Variable residues (grey), perfectly conserved residues (green) and coordinated RNA (red).