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Schematic representation of glycopeptide antibiotic biosynthesis by a combination of non-ribosomal peptide synthesis and Cytochrome P450-mediated (Oxy enzyme mediated) side chain crosslinking; examples of a type I (vancomycin) and type IV (teicoplanin) glycopeptide antibiotic aglycone are shown.: Non-ribosomal peptide synthetase domains are indicated using the following nomenclature. A (adenylation), PCP (peptidyl carrier protein), C (condensation), E (epimerisation), X (P450 recruitment) and TE (thioesterase). Incorporated amino acids are indicated above the modules: Hpg (4-hydroxyphenylglycine), Dpg (3,5-dihydroxyphenylglycine). Oxy enzyme crosslinking is shown for the teicoplanin precursor peptide.
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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...
Citations
... [4][5][6]17,[28][29][30][31][32][33][34] OxyE is present in type III-IV GPAs and installs the F-O-G ring between residues 1 and 3, with this occurring after OxyB activity but before that of OxyA. [26,35] A comparable OxyE enzyme has been identified in corbomycin biosynthesis, although the order of activity in this case is as yet unclear. [11] This pathway is further complicated by the possibility of type V OxyC enzymes to install both links normally installed by OxyB and OxyC in type I-IV GPAs, [14] as well as evidence from in-vivo studies of complex mixtures of crosslinked peptides resulting from Oxy deletion. ...
The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell‐wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II‐binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II‐binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C‐terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C‐terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.
... Bicyclic diaryl ether linkages were found in the structures of glycopeptide antibiotics such as vancomycin, teicoplanin, and A47934. These ether linkages between two phenol rings were constructed by cytochrome P450 OxyB enzyme [53]. EpcH, cytochrome P450 in the BGC of epoxinnamide, showed 35% of sequence identity with OxyB in the BGC of teicoplanin [54]. ...
Cinnamoyl-containing nonribosomal peptides (CCNPs) form a unique family of actinobacterial secondary metabolites and display various biological activities. A new CCNP named epoxinnamide (1) was discovered from intertidal mudflat-derived Streptomyces sp. OID44. The structure of 1 was determined by the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data along with a mass spectrum. The absolute configuration of 1 was assigned by the combination of advanced Marfey’s method, 3JHH and rotating-frame overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis. The putative biosynthetic pathway of epoxinnamide (1) was identified through the whole-genome sequencing of Streptomyces sp. OID44. In particular, the thioesterase domain in the nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster was proposed as a bifunctional enzyme, which catalyzes both epimerization and macrocyclization. Epoxinnamide (1) induced quinone reductase (QR) activity in murine Hepa-1c1c7 cells by 1.6-fold at 5 μM. It also exhibited effective antiangiogenesis activity in human umbilical vein endothelial cells (IC50 = 13.4 μM).
... As such, X domains are responsible for the trans-activation of tailoring enzymes [72]. Three cytochrome p450 enzymes for vancomycin, and four for teicoplanin, are sequentially recruited by the dedicated X domain in their respective biosynthetic lines to catalyze amino acid sidechains cross-linkings resulting in the formation of three or four, respectively, macrocycles and therefore providing aglycone molecules, which already possess the rigid three-dimensional structure that is responsible for the antibiotic activity of the GPAs [72,74,75]. A cristallography study showed that during the biosynthesis of teicoplanin, the multiple cytochrome p450 enzymes required for the macrocyclizations all interact in a sequential manner according to the cyclization status of the nascent peptide, with the same site on the X domain surface for which they compete. ...
Nonribosomal peptides are microbial secondary metabolites exhibiting a tremendous structural diversity and a broad range of biological activities useful in the medical and agro-ecological fields. They are built up by huge multimodular enzymes called nonribosomal peptide synthetases. These synthetases are organized in modules constituted of adenylation, thiolation, and condensation core domains. As such, each module governs, according to the collinearity rule, the incorporation of a monomer within the growing peptide. The release of the peptide from the assembly chain is finally performed by a terminal core thioesterase domain. Secondary domains with modifying catalytic activities such as epimerization or methylation are sometimes included in the assembly lines as supplementary domains. This assembly line structure is analyzed by bioinformatics tools to predict the sequence and structure of the final peptides according to the sequence of the corresponding synthetases. However, a constantly expanding literature unravels new examples of nonribosomal synthetases exhibiting very rare domains and noncanonical organizations of domains and modules, leading to several amazing strategies developed by microorganisms to synthesize nonribosomal peptides. In this review, through several examples, we aim at highlighting these noncanonical pathways in order for the readers to perceive their complexity.
... In vivo gene disruption experiments have shown that the oxidative crosslinking reactions are catalysed by a cascade of trans-acting cytochrome P450 monooxygenases (known as Oxy enzymes) while the heptapeptide substrate is still attached to the NRPS assembly line. [131][132][133][134][135][136][137][138] A comprehensive study by Haslinger and Peschke et al. revealed that an atypical C domain within the final module of the NRPS, called the X domain, is reponsible for recruiting and aligning the oxygenase enzymes towards the adjacent PCP domain (Fig. 15). 127 Structural characterization of the teicoplanin X domain by X-ray crystallography showed that it adopts the same overall fold as canonical C domains, but harbours a modified HRxxxDD active site motif. ...
... 127 Subsequent in vitro kinetic experiments revealed that the different oxygenase enzymes continuously compete for the same interaction interface on the X domain and are able to 'scan' the cyclization state of the peptidyl thioester intermediate. [137][138][139] Recognition of the correct substrate leads to a conformational change in the enzymes that enhances their affinity for the substrate and initiates catalysis. 139 X domains as enzyme recruitment platforms are therefore yet another testament to the remarkable functional versatility of C domains. ...
Nonribosomal peptide synthetases are remarkable molecular machines that produce a wide range of structurally complex peptide natural products with important applications in medicine and agriculture. Condensation domains play a central role in these biosynthetic pathways by catalysing amide bond formation between various aminoacyl substrates. In recent years, however, it has become increasingly clear that the catalytic repertoire of C domains extends far beyond conventional peptide bond formation. C domains have been shown to perform highly diverse functions during nonribosomal peptide assembly, such as β-lactam formation, dehydration, hydrolysis, chain length control, cycloaddition, Pictet–Spengler cyclization, Dieckmann condensation and recruitment of auxiliary enzymes. In this review, a comprehensive overview of the multifaceted role of C domains in the biosynthesis of specialized metabolites in bacteria and fungi is presented. Different perspectives are also offered on how the exceptional functional versatility of C domains may be exploited for bioengineering approaches to expand the chemical diversity of nonribosomal peptides and other natural products.
... Comparison of synthetic tripeptide standards ( Fig. 10D) with the products of Tcp9 + Tcp10 incubations using both L-Hpg, L-Cl-Tyr and L-Dpg, and D-Hpg, L-Cl-Tyr and L-Dpg indicated that D/D/(L/D) products were produced in both assays (Fig. 10B, purple and red traces). The double peaks in the tripeptides produced by the assay are the result of epimerisation of C-terminal Dpg residues upon cleavage of the thioester linking them to the carrier protein domain, which has been observed previously with such peptides [4,[35][36][37][38]. Here again, the incorporation of D-Hpg in the second experiment clearly differentiates the D/D/(L/D) product produced here from the possible formation of L/D(L/D) tripeptides, which have significantly longer retention times. ...
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.
... This domain is a descendent of a C-domain from the ancestral proto-GPA NRPS (Peschke et al. 2016a). This recruits cytochrome P450 oxygenases involved in cyclization of the NRPS-bound heptapeptide (Haslinger et al. 2015;Peschke et al. 2016b), thereby providing an evolutionary rationale for retention of this catalytically inert domain. ...
Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.
... Due to the clinical utility of GPAs and the challenges associated with the synthesis of these complex molecules, significant research efforts have been made to understand the cyclisation process of GPAs from both an in vivo 3,10,11 and in vitro standpoint 9,[12][13][14][15] . These studies have revealed that each Oxy enzyme is responsible for the installation of one crosslink at the heptapeptide stage (Fig. 1b) and that a specific order of activity exists (OxyB, (±OxyE), OxyA, OxyC). ...
Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.
... 23 The X-domain, found in the nal NRPS module of all GPA producing assemblies, is an example of a modied C-domain and the only other reported example of a C/E type domain immediately prior to a terminal thioesterase domain along with the penicillin producing d-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthase. 23,24 Whilst in vitro results have been supportive of the X-domain playing a role in the complete enzymatic crosslinking cascade introduced at the heptapeptide stage (and hence on the nal NRPS module), 23,[25][26][27][28] in vivo experiments provide a different hypothesis favouring hexapeptide cyclisation for all steps before that of the nal AB ring Biosynthetic scheme for the glycopeptide antibiotics (GPAs), exemplified for teicoplanin (type-IV GPA, upper panel) as well as related GPA structures relevant for this work actinoidin (type-II GPA, lower left) and balhimycin (type-I GPA lower right). Type-III GPAs possess the same core peptide sequence as type-IV GPAs. ...
Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many...
... The essential role of the X-domain in GPA crosslinking has been implied in a number of in vivo [118][119][120] and more recently proven by in vitro experiments, where the use of X-domain containing constructs have allowed the characterisation of both OxyE and OxyA enzymes for the rst time. 108,121,[123][124][125] Denitive evidence that the X-domain was indeed a binding platform for the Oxy enzymes came with the structure of the complex between the X-domain and OxyB from the teicoplanin NRPS assembly line. 121 In this structure, as anticipated, the fold of the X-domain resembled that of a C/Edomain, albeit with insertions that blocked the tunnel usually occupied by the acceptor PCP substrate. ...
Covering: up to July 2018
Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. By decoupling peptide synthesis from the ribosome, NRPS assembly lines are able to access a significant pool of amino acid monomers for peptide synthesis. This is combined with a modular protein architecture that allows for great variation in stereochemistry, peptide length, cyclisation state and further modifications. The architecture of NRPS assembly lines relies upon a repetitive set of catalytic domains, which are organised into modules responsible for amino acid incorporation. Central to NRPS-mediated biosynthesis is the carrier protein (CP) domain, to which all intermediates following initial monomer activation are bound during peptide synthesis up until the final handover to the thioesterase domain that cleaves the mature peptide from the NRPS. This mechanism makes understanding the protein–protein interactions that occur between different NRPS domains during peptide biosynthesis of crucial importance to understanding overall NRPS function. This endeavour is also highly challenging due to the inherent flexibility and dynamics of NRPS systems. In this review, we present the current state of understanding of the protein–protein interactions that govern NRPS-mediated biosynthesis, with a focus on insights gained from structural studies relating to CP domain interactions within these impressive peptide assembly lines.
... 254 The X-domain is also required in the majority of cases to support efficient P450catalysed crosslinking in vitro, with this requirement strictly enforced for OxyA and OxyE that act aer OxyB. 243,255,256,[258][259][260][261][262][263] Complestatin 264 and kistamicin 265 are structurally similar to GPAs, and also display several oxidative crosslinks whose generation has been ascribed to P450 enzymes, i.e. NRPS-bound modication with X-domain mediated P450 recruitment. ...
Covering: 2000 up to 2018
The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C–H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways.
