Clara Brieke

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450–X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro—in particular the installation of the second crosslink in GPA biosynthesis—occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.

Reversible photoswitching of RNA hybridization at room temperature with an azobenzene C-nucleoside.

Photoregulation of RNA remains a challenging task as the introduction of a photoswitch entails changes in the shape and the stability of the duplex that strongly depend on the chosen linker strategy. Herein, the influence of a novel nucleosidic linker moiety on the photoregulation efficiency of azobenzene is investigated. To this purpose, two azobenzene C-nucleosides were stereoselectively synthesized, characterized, and incorporated into RNA oligonucleotides. Spectroscopic characterization revealed a reversible and fast switching process, even at 20 °C, and a high thermal stability of the respective cis isomers. The photoregulation efficiency of RNA duplexes upon trans-to-cis isomerization was investigated by using melting point studies and compared with the known D-threoninol-based azobenzene system, revealing a photoswitching amplitude of the new residues exceeding 90 % even at room temperature. Structural changes in the duplexes upon photoisomerization were investigated by using MM/MD calculations. The excellent photoswitching performance at room temperature and the high thermal stability make these new azobenzene residues promising candidates for in-vivo and nanoarchitecture photoregulation applications of RNA.

Cytochrome P450 OxyBtei Catalyzes the First Phenolic Coupling Step in Teicoplanin Biosynthesis

Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as “Oxy enzymes” play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non‐ribosomal antibiotic precursor peptide while it remains bound to the non‐ribosomal peptide synthetase (NRPS); this crosslinking secures the three‐dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei, the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS‐bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well‐studied vancomycin homologue.

Sequential In Vitro Cyclization by Cytochrome P450 Enzymes of Glycopeptide Antibiotic Precursors Bearing the X-Domain from Nonribosomal Peptide Biosynthesis.

The biosynthesis of the glycopeptide antibiotics, which include vancomycin and teicoplanin, relies on the interplay between the peptide-producing non-ribosomal peptide synthetase (NRPS) and Cytochrome P450 enzymes (P450s) that catalyze side-chain crosslinking of the peptide. We demonstrate that sequential in vitro P450-catalyzed cyclization of peptide substrates is enabled by the use of an NRPS peptide carrier protein (PCP)-X di-domain as a P450 recruitment platform. This study reveals that whilst the precursor peptide sequence influences the installation of the second crosslink by the P450 OxyAtei , activity is not restricted to the native teicoplanin peptide. Initial peptide cyclization is possible with teicoplanin and vancomycin OxyB homologues, and the latter displays excellent activity with all substrate combinations tested. By using non-natural X-domain substrates, bicyclization of hexapeptides was also shown, which demonstrates the utility of this method for the cyclization of varied peptide substrates in vitro.

A Facile Fmoc Solid Phase Synthesis Strategy To Access Epimerization-Prone Biosynthetic Intermediates of Glycopeptide Antibiotics

A rapid protocol based on Fmoc-chemistry for the solid phase peptide synthesis of vancomycin- and teicoplanin-type peptides is described. Epimerization of highly racemization-prone arlyglycine derivatives is suppressed through optimized Fmoc-deprotection and coupling conditions. Starting from easily accessible Fmoc-protected amino acids, this strategy enables the enantioselective synthesis of peptides corresponding to intermediates found in vancomycin and teicoplanin biosynthesis with excellent purity and in high yields (38%-71%).

Regulation of the P450 Oxygenation Cascade Involved in Glycopeptide Antibiotic Biosynthesis

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis.

Rapid access to glycopeptide antibiotic precursor peptides coupled with cytochrome P450-mediated catalysis: towards a biomimetic synthesis of glycopeptide antibiotics

Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective in vitro characterization of the crucial later steps of the biosynthesis, facile access to a wide range of substrate peptides as their Coenzyme A (CoA) conjugates is essential. Here we report the development of a rapid route to glycopeptide precursor CoA conjugates that affords both high yields and excellent purities. This synthesis route is applicable to the synthesis of peptide CoA-conjugates containing racemization-prone arylglycine residues: such residues are hallmarks of non-ribosomal peptide synthesis and have previously been inaccessible to peptide synthesis using Fmoc-type chemistry. We have applied this route to generate glycopeptide precursor peptides in their carrier protein-bound form as substrates to explore the specificity of the first oxygenase enzyme from vancomycin biosynthesis (OxyBvan). Our results indicate that OxyBvan is a highly promiscuous catalyst for phenolic coupling of diverse glycopeptide precursors that accepts multiple carrier protein substrates, even on carrier protein domains from alternate glycopeptide biosynthetic machineries. These results represent the first important steps in the development of an in vitro biomimetic synthesis of modified glycopeptide aglycones.

Azobenzene C-Nucleosides for Photocontrolled Hybridization of DNA at Room Temperature.

Herein, we report the reversible light-regulated destabilization of DNA duplexes by using azobenzene C-nucleoside photoswitches. The incorporation of two different azobenzene residues into DNA and their photoswitching properties are described. These new residues demonstrate a photoinduced destabilization effect comparable to the widely applied D-threoninol-linked azobenzene switch, which is currently the benchmark. The photoswitches presented herein show excellent photoswitching efficiencies in DNA duplexes - even at room temperature - which are superior to commonly used azobenzene-based nucleic acid photoswitches. In addition, these photoswitching residues exhibit high thermal stability and excellent fatigue resistance, thus rendering them one of the most efficient candidates for the regulation of duplex stability with light.

F-O-G ring formation in glycopeptide antibiotic biosynthesis is catalysed by OxyE

The glycopeptide antibiotics are peptide-based natural products with impressive antibiotic function that derives from their unique three-dimensional structure. Biosynthesis of the glycopeptide antibiotics centres of the combination of peptide synthesis, mediated by a non-ribosomal peptide synthetase, and the crosslinking of aromatic side chains of the peptide, mediated by the action of a cascade of Cytochrome P450s. Here, we report the first example of in vitro activity of OxyE, which catalyses the F-O-G ring formation reaction in teicoplanin biosynthesis. OxyE was found to only act after an initial C-O-D crosslink is installed by OxyB and to require an interaction with the unique NRPS domain from glycopeptide antibiotic – the X-domain – in order to display catalytic activity. We could demonstrate that OxyE displays limited stereoselectivity for the peptide, which mirrors the results from OxyB-catalysed turnover and is in sharp contrast to OxyA. Furthermore, we show that activity of a three-enzyme cascade (OxyB/OxyA/OxyE) in generating tricyclic glycopeptide antibiotic peptides depends upon the order of addition of the OxyA and OxyE enzymes to the reaction. This work demonstrates that complex enzymatic cascades from glycopeptide antibiotic biosynthesis can be reconstituted in vitro and provides new insights into the biosynthesis of these important antibiotics.