Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi  ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.

Halogenase enzymes involved in glycopeptide antibiotic biosynthesis accept aminoacyl-carrier protein substrates.

The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.