Torsten Stachelhaus

Structural basis for the activation of phenylalanine in the non‐ribosomal biosynthesis of gramicidin S

The non‐ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of ∼60 kDa which carry out ATP‐dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl‐CoA ligases. We have determined the crystal structure of the N‐terminal adenylation subunit in a complex with AMP and L‐phenylalanine to 1.9 Å resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94° occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the α‐amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate‐forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.

Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain.

Recently, considerable insight has been gained into the modular organization and catalytic properties of nonribosomal peptide synthetases. However, molecular and biochemical aspects of the condensation of two aminoacyl substrates or a peptidyl and an aminoacyl substrate, leading to the formation of a peptide bond, have remained essentially impenetrable. To investigate this crucial part of nonribosomal peptide synthesis, an in vitro assay for a dipeptide formation was developed. Two recombinant holomodules, GrsA (PheATE), providing d-Phe, and a C-terminally truncated TycB, corresponding to the first, l-Pro-incorporating module (ProCAT), were investigated. Upon combination of the two aminoacylated modules, a fast reaction is observed, due to the formation of the linear dipeptided-Phe-l-Pro-S-enzyme on ProCAT, followed by a noncatalyzed release of the dipeptide from the enzyme. The liberated product was identified by TLC, high pressure liquid chromatography-mass spectrometry, 1H and 13C NMR, and comparison with a chemically synthesized standard to be the expectedd-Phe-l-Pro diketopiperazine. Further minimization of the two modules was not possible without a loss of transfer activity. Likewise, a mutation in a proposed active-site motif (HHXXXDG) of the condensation domain giving ProCAT(H147V), abolished the condensation reaction. These results strongly suggest the condensation domain to be involved in the catalysis of nonribosomal peptide bond formation with the histidine 147 playing a catalytic role.

Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

BACKGROUND Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.

In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

ABSTRACT Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide d-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4′-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. d-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of d-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

Engineered biosynthesis of peptide antibiotics

In certain bacteria and filamentous fungi, a wide variety of bioactive peptides are produced non-ribosomally on large protein templates, called peptide synthetases. Recently, significant progress has been made towards understanding the modular arrangement of these complex multifunctional enzymes and the mechanisms by which they generate their corresponding peptide products. It has now been established that the synthesis of bioactive peptides and the specification of their sequence are brought about by a protein template that contains the appropriate number and the correct order of activating units (domains). These advances have enabled the development of a technique that permits the construction of hybrid genes encoding peptide synthetases with specifically altered substrate specificities. A programmed alteration within the primary structure of a peptide antibiotic is achieved by the substitution of an amino acid-activating domain in the corresponding protein template at the genetic level by a two-step recombination method. It utilizes successive gene disruption and reconstitution and demonstrates, for the first time, the potential of genetic engineering in the biosynthesis of novel peptide antibiotics. Many organisms, for instance those that cause diseases like tuberculosis and pneumonia, have evolved potent mechanisms of drug resistance. Therefore, the targeted engineering of peptide antibiotics could be one potential strategy for the development of novel drugs that overcome this resistance.