Figure - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
Structural overview of glycopeptide antibiotics. (a) Order of cyclization reactions and enzymes involved during the biosynthesis of A47934. Altered GPA structures of (b) bromobalhimycin, produced by supplementation of the media with bromine salts, and (c) an octapeptide, produced by an engineered NRPS Amycolatopsis balhimycina mutant.
Source publication
The importance of Cytochrome P450-catalyzed modifications of natural products produced by non-ribosomal peptide synthetase machineries is most apparent during glycopeptide antibiotic biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the peptide backbone of these clinically relevant antibiotics. These cycliz...
Citations
... [1,2] Central to this is the role of a nonribosomal peptide synthetase (NRPS) assembly line that generates the linear peptide core, [3] and the subsequent activity of a cascade of cytochrome P450 (Oxy) enzymes [4][5][6] that perform the signature sidechain crosslinking seen in GPAs ( Figure 1). [7,8] Whilst most reported GPAs, including both the clinical examples of GPAs (vancomycin, teicoplanin) as well as second generation semi-synthetic GPAs (oritavancin, dalbavancin) consist of heptapeptide backbones bearing three or four side-chain crosslinks that target peptidoglycan biosynthesis by inhibiting the incorporation of lipid II, a major effort from the Wright group has shown the diversity of GPAs extends beyond lipid II-binding GPAs into the so-called type V GPAs. [9][10][11][12] Type V GPAs, exemplified by complestatin, [13] kistamicin [4] and more recently corbomycin, [11] show significant differences in structure to lipid II-binding GPAs, including a lack of glycosylation, altered peptide backbones (including extension beyond heptapeptides) and different crosslinking patterns within their aglycones ( Figure 1). ...
... [12] Despite these differences, the key biosynthetic and structural feature of GPA biosynthesis-the complex, Oxy-mediated crosslinking of the linear peptide-is maintained across all GPAs studied to date. [8,[15][16][17] Indeed, a previous investigation of the crosslinking cascade found in the biosynthesis of the type V GPA kistamicin showed that this process remained linked to peptide biosynthesis on the NRPS assembly line through the recruitment of the Oxy enzymes via an X-domain. [14] This domain-unique to GPA biosynthesis-interacts with the Oxy enzymes and delivers the adjacent peptidyl carrier protein (PCP)-bound peptide substrate to these cyclisation enzymes and appears conserved across all GPAs. ...
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.
... 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. ...
... The X domain-cytochrome p450 enzymes interactions take place at a micromolar range and occur mainly through hydrogen bonds and salt bridges [72]. Notably, unlike other cytochrome p450 enzymes, the cytochrome p450 enzymes recruited by a GPA X domain bear a conserved Pro-Arg-Asp-Asp motif in the F-helix [75]. Finally, it is worthy to note that the X domain, together with a 120 amino acid extension close to the Te domain, is thought to originally represent an additional module in an ancestor GPA-producing NRPS [34]. ...
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.
... Glycopeptide antibiotics (GPAs), such as vancomycin (13), have been used clinically to treat serious infections caused by Gram-positive bacteria for decades (Levine, 2006, Kahne et al., 2005, Yim et al., 2014. Structurally, vancomycin (13) The linear heptapeptide backbone (8) is assembled by non-ribosomal peptide synthetase (NRPS) and contains Asn, Leu, and five non-proteinogenic aromatic amino acids (Yim et al., 2014, Haslinger et al., 2015, Peschke et al., 2016. The aromatic side-chain crosslinking of the heptapeptide precursor 8 is catalyzed by three P450 enzymes (OxyA, OxyB, and OxyC) with a strict order, featuring multiple C−O and C−C coupling steps (Bischoff et al., 2001, Stegmann et al., 2006 ...
Radical cyclizations are essential reactions in the biosynthesis of secondary metabolites and the chemical synthesis of societally valuable molecules. In this review, we highlight the general mechanisms utilized in biocatalytic radical cyclizations. We specifically highlight cytochrome P450 monooxygenases (P450s) involved in the biosynthesis of mycocyclosin and vancomycin, non-heme iron- and α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) used in the biosynthesis of kainic acid, scopolamine, and isopenicillin N, and radical S-adenosylmethionine (SAM) enzymes that facilitate the biosynthesis of oxetanocin A, menaquinone, and F420. Beyond natural mechanisms, we also examine repurposed flavin-dependent ‘ene’-reductases (ERED) for non-natural radical cyclization. Overall, these general mechanisms underscore the opportunity for enzymes to augment and enhance the synthesis of complex molecules using radical mechanisms.
... [1] The complex structure of GPAs, which includes multiple crosslinks between the side chains of aromatic residues within the peptide core, is a fascinating example of the power of biosynthesis to produce novel bioactive structures. The biosynthesis of GPAs, which links nonribosomal peptide synthesis [2] of the linear peptide precursor to an oxidative cyclisation cascade to produce the crosslinked peptide aglycone, [3] is further complemented by significant diversity in post-peptide synthesis modifications ( Figure 1). [1] The synthesis of GPAs has been the subject of intense research that has delivered impressive efforts in compound redesign to overcome bacterial resistance to this compound class. ...
... A crystal structure of a substrate bound Oxy enzyme would also be of great benefit for understanding the selectivity of these enzymes, although this remains highly challenging given the transience of the Oxy/X complex. [3,62] Linking this work with re-engineering the peptide-producing NRPS is also a clear priority if we are to be able to access modified GPA peptides at a scale relevant for clinical application. [40,63] Taken together, these are exciting times for GPA research, with the in vitro reconstitution of the GPA cyclisation cascade providing a major advance in our understanding of this fascinating biosynthetic pathway . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 ...
The glycopeptide antibiotics (GPAs) are a fascinating example of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450‐mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X‐domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.
... 31,32 Indeed, crystal structure analysis indicated that an additional X-domain or PCP binding site is indispensable for recruiting stand-alone P450s to the NRPS for ether bond formation or b-hydroxylation in glycopeptide biosynthesis. 33,34 Different to the more common stand-alone P450s in NRPS biosynthesis, the bsm cluster encodes a monomodular NRPS (P450-A-T) with an extra cytochrome P450 monooxygenase domain fused at the N-terminal. As a domain, a P450 monooxygenase has never been a part of a NRPS megaenzyme. ...
Nonribosomal peptides (NRPs) that are synthesized by modula megaenzymes known as nonribosomal peptide synthetases (NRPSs) are a rich source for drug discovery. Targeting unusual NRPS architechture, we discovered an unusual biosynthetic gene cluster (bsm) from Streptomyces sp. 120454 and identified that it was responsible for the biosynthesis of a series of novel linear peptides, bosamycins. The bsm gene cluster contains a unique monomodule NRPS, BsmF, that contains a cytochrome P450 domain at the N-terminal. BsmF (P450+A+T) can selectively activate tyrosine by its adenylation (A) domain and load it onto the thiolation (T) domain, then hydroxylate tyrosine to form 5-OH tyrosine by the P450 domain. We demonstrated a NRPS assembly line for the formation of bosamycins by genetic, biochemical analysis and heterologous expression. Our work reveals a genome mining strategy targeting unique NRPS domain for the discovery of novel NRPs.
... [30][31][32][33][34][35] OxyA introduces the same modification on the opposite side of the molecule, forging an E-O-D bond. [36][37][38] Finally, OxyC catalyzes the formation of a C─C bond between 3,5-dihydroxyphenylglcyine (Dpg) and Hpg to generate the AB motif ( Figure 1). [39][40][41][42] These intramolecular cyclizations create a rigid cup-shaped structure capable of tight binding to the N-acyl-D-Ala-D-Ala dipeptide at the terminus of peptidoglycan strands, thereby preventing peptidoglycan polymerization and weakening bacterial cell walls. ...
Vancomycin is one of the most important clinical antibiotics in the fight against infectious disease. Its biological activity relies on three aromatic crosslinks, which create a cup-shaped topology and allow tight binding to nascent peptidoglycan chains. The cytochrome P450 enzymes OxyB, OxyA and OxyC have been shown to introduce these synthetically challenging aromatic linkages. The ability to utilize the P450 enzymes in a chemo-enzymatic scheme to generate vancomycin derivatives is appealing but requires a thorough understanding of their reactivities and mechanisms. Herein, we systematically explore the scope of OxyB biocatalysis and report installation of diverse diaryl ether and biaryl crosslinks with varying macrocycle sizes and compositions, when the enzyme is presented with modified vancomycin precursor peptides. The structures of the resulting products were determined using 1D/2D NMR, HR-MS, tandem HR-MS, isotopic labeling, as well as UV-vis absorption and fluorescence emission spectroscopies. An exploration of the biological activities of these alternative OxyB products surprisingly revealed antifungal properties. Taking advantage of the promiscuity of OxyB, we chemo-enzymatically generated the first vancomycin aglycone variant containing an expanded macrocycle. Mechanistic implications for OxyB and future directions for creating vancomycin variant libraries are discussed.
... A unique feature of GPA NRPSs is the X-domain of module 7. 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.
... All of these stand-alone enzymes produce L-erythro (2S, 3R) or D-threo (2R, 3R) β-OHAsp, requiring opposite stereospecificity of β-hydroxylation relative to IβH Asp domains (Table 1 and Fig. 6). Remarkably, the 2 drivers of residue specificity we propose here (i.e., the interface domain and the T E -type thiolation domain; Fig. 3) parallel selectivity seen in the cytochrome P450 family of enzymes involved in glycopeptide antibiotic and skyllamycin biosyntheses (60). These similarities, as well as the stereochemistry of β-hydroxylation, bear further consideration. ...
... Cytochrome P450 sky of skyllamycin (SI Appendix, Fig. S2) biosynthesis similarly selects residues for β-hydroxylation by interacting only with specific T domains, as determined by X-ray crystallography (65). In contrast to the T E domain, even noninteracting T domains within the skyllamycin pathway contain the key residues for P450 sky interaction, and selectivity is believed to arise from minor changes in T domain tertiary structure that disrupt the NRPS/P450 sky interface (60,65). TβH Asp enzymes may similarly select modules for binding based on subtle structural changes, undetectable in the primary sequence of the NRPS. ...
Significance
Bacteria produce siderophores to sequester iron(III). Genome mining of nonribosomal peptide synthetases predicts partial structures of peptidic siderophores; however, current tools cannot reliably predict which aspartate and histidine residues will be hydroxylated to form bidentate chelating groups, nor the resulting stereochemistry. We identified 2 functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases in siderophore biosynthetic gene clusters and one type of histidyl β-hydroxylase. Stand-alone genes encode one class of aspartyl β-hydroxylases and the histidyl β-hydroxylases, while the second class of aspartyl β-hydroxylases is encoded within a domain of a nonribosomal peptide synthetase (NRPS) gene. Each aspartyl β-hydroxylase subtype effects distinct diastereoselectivity. Mapping the β-OHAsp diastereomers in siderophores to the phylogenetic tree of β-hydroxylases enables prediction of β-OHAsp stereochemistry in silico.
... While OxyB from the vancomycin pathway (OxyBvan) exhibits a rather broad substrate scope and is capable of accepting a variety of peptides tethered to peptidyl carrier proteins (PCPs), the homolog from the teicoplanin pathway (OxyBtei; 74% sequence identity) is much more discriminating and is unable to accept nonnative peptide substrates. Moreover, OxyBtei activity is highly dependent on an auxiliary nonribosomal peptide synthetase (NRPS) domain, which serves as a recruitment platform for the P450s that catalyze aryl and phenolic coupling reactions in the biosynthesis of glycopeptide antibiotics (53,54). In the study, the BC loop and/or the F and G helices (including the FG loop) were transplanted from OxyBvan to OxyBtei, and the activities of the resulting chimeras were assessed (52). ...
Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99 Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes.
... 15 The crosslinking cascade in GPA biosynthesis has been shown to depend on recruitment of external cytochrome P450 monooxygenases through the conserved X-domain within the nal NRPS module, a process unique to GPAs. 16,17 The process controlling the modication of amino acid residues within the peptide, however, is less clear. Different mechanisms also exist for incorporation of specic amino acid modications during NRPS-mediated biosynthesis of vancomycin and teicoplanintype GPAs. ...
Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear. Here, we have characterised the incorporation of the non-proteinogenic amino acid 3-chloro-β-hydroxytyrosine during glycopeptide antibiotic (GPA) biosynthesis. Our results demonstrate that the modification of this residue by trans-acting enzymes is controlled by the selectivity of the upstream condensation domain responsible for peptide synthesis. A proofreading thioesterase works together with this process to ensure that effective peptide biosynthesis proceeds even when the selectivity of key amino acid activation domains within the NRPS is low. Furthermore, the exchange of condensation domains with altered amino acid specificities allows the modification of such residues within NRPS biosynthesis to be controlled, which will doubtless prove important for reengineering of these assembly lines. Taken together, our results indicate the importance of the complex interplay of NRPS domains and trans-acting enzymes to ensure effective GPA biosynthesis, and in doing so reveals a process that is mechanistically comparable to the hydrolytic proofreading function of tRNA synthetases in ribosomal protein synthesis.
