Structural elements of an NRPS cyclization domain and its intermodule docking domain.

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.

Biosynthesis of the Thiopeptins and Identification of an F420H2-Dependent Dehydropiperidine Reductase.

Thiopeptins are highly decorated thiopeptide antibiotics similar in structure to thiostrepton A and harbor two unusual features. All thiopeptins contain a thioamide, a rare moiety among natural products, and a subset of thiopeptins present with a piperidine in the core macrocycle rather than the more oxidated dehydropiperidine or pyridine rings typically observed in the thiopeptides. Here, we report the identification of the thiopeptin biosynthetic gene ( tpn) cluster in Streptomyces tateyamensis and the gene product, TpnL, which shows sequence similarity to (deaza)flavin-dependent oxidoreductases. Heterologous expression of TpnL in the thiostrepton A producer Streptomyces laurentii led to the production of a piperidine-containing analogue. Binding studies revealed that TpnL preferentially binds the deazaflavin cofactor coenzyme F420, and in vitro reconstitution of TpnL activity confirmed that this enzyme is an F420H2-dependent dehydropiperidine reductase. The identification of TpnL and its activity establishes the basis for the piperidine-containing series a thiopeptides, one of the five main structural groups of this diverse family of antibiotics.

Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase.

TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Recent advances in thiopeptide antibiotic biosynthesis.

Thiopeptides, or thiazolylpeptides, are a family of highly modified peptide antibiotics first discovered several decades ago. Dozens of thiopeptides have since been identified, but, until recently, the biosynthetic genes responsible for their production remained elusive. The biosynthetic systems for a handful of thiopeptide metabolites were identified in the first portion of 2009. The surprising finding that these metabolites arise from the enzymatic tailoring of a simple, linear, ribosomally-synthesized precursor peptide led to a renewed appreciation of the architectural complexity accessible by posttranslational modification. This recent progress toward understanding thiopeptide antibiotic biosynthesis benefits the discovery of novel thiopeptides by either directed screening techniques or by mining available microbial genome sequences. Furthermore, access to the biosynthetic machinery now opens an avenue to the biosynthetic engineering of thiopeptide analogs. This Highlight discusses the genetic and biochemical insights revealed by these initial reports of the biosynthetic gene clusters for thiopeptide metabolites.

Intermittent Parathyroid Hormone Accelerates Stress Fracture Healing More Effectively Following Cessation of Bisphosphonate Treatment.

Parathyroid hormone (PTH) and bisphosphonates (BPs), including alendronate (ALN), have opposing effects on bone dynamics. The extent to which PTH remains effective in the treatment of stress fracture (SFx) in the presence of an ongoing BP treatment has not been tested. SFx was induced in 150 female Wistar rats, divided into five equal groups (n = 30). All rats were pretreated with ALN (1 µg/kg-1/day-1) for 14 days prior to SFx induction, followed by ALN cessation or continuation for the duration of the experiment; this was combined with daily PTH (8 µg/100 g-1/day-1) on SFx induction for 14 days, followed by cessation or continuation of ALN after SFx induction or an equivalent vehicle as a control. Ulnas were examined 2 weeks or 6 weeks following SFx. Two toluidine blue- and two tartrate-resistant acid phosphatase-stained sections were examined for histomorphometric analysis using Osteomeasure software. There was a significant interaction between the effects of time and treatment type on the woven bone width and apposition rate, as well as an improvement in the woven bone architecture. However, woven bone variables remained unaffected by the cessation or continuation of ALN. Cessation of ALN increased osteoclast number when compared with the ALN-PTH continuation group (p = 0.006), and vehicle (p = 0.024) after 2 weeks. There was a significant interaction between the effects of time and treatment type on the number of osteoclasts per unit BMU area and length. The number of osteoclasts per unit BMU area and length was significantly greater in ALN cessation groups. It was concluded that intermittent short-duration iPTH treatment effectively increased remodeling of SFx with a concurrent BP treatment, provided that BP was ceased at the time of SFx. Our results could help develop shorter iPTH treatment protocols for the clinical management of SFxs and guide clinical decision-making to cease BP treatment in cases of SFx. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Thiostrepton biosynthesis: prototype for a new family of bacteriocins.

Thiopeptide antibiotics are a group of highly modified peptide metabolites. The defining scaffold for the thiopeptides is a macrocycle containing a dehydropiperidine or pyridine ring, dehydrated amino acids, and multiple thiazole or oxazole rings. Some members of the thiopeptides, such as thiostrepton, also contain either a quinaldic acid or indolic acid substituent derived from tryptophan. Although the amino acid precursors of these metabolites are well-established, the biogenesis of these complex peptides has remained elusive. Whole-genome scanning of Streptomyces laurentii permitted identification of a thiostrepton prepeptide, TsrA, and involvement of TsrA in thiostrepton biosynthesis was confirmed by mutagenesis. A gene cluster responsible for thiostrepton biosynthesis is reported, and the encoded gene products are discussed. The disruption of a gene encoding an amidotransferase, tsrT, led to the loss of thiostrepton production and the detection of a new metabolite, contributing further support to the identification of the tsr cluster. The tsr locus also appears to possess the gene products needed to convert tryptophan to the quinaldic acid moiety, and an aminotransferase was found to catalyze an early step in this pathway. This work establishes that the thiopeptides are a type of bacteriocin, a family of genetically encoded antimicrobial peptides, and are subjected to extensive posttranslational modification during maturation of the prepeptide.

Analysis of the indanomycin biosynthetic gene cluster from Streptomyces antibioticus NRRL 8167.

Metabolites that harbor a core indane scaffold are found to have diverse biological properties. Indanomycin and related pyrroloketoindanes are ionophores and have demonstrated antiparasitic, insecticidal, and antibacterial activities. To understand the biochemical mechanisms guiding formation of the central indane ring, the biosynthetic gene cluster for indanomycin was identified from Streptomyces antibioticus NRRL 8167 and sequenced to approximately 80 kb; this revealed five genes encoding subunits of a polyketide synthase (PKS) and 18 other open reading frames. The involvement of this cluster in indanomycin biosynthesis was confirmed by deletion mutagenesis. The indanomycin PKS lacks the expected thioesterase at the carboxy terminus of the final module, and instead appears to house an incomplete module containing an unusual cyclase domain. These findings now enable additional detailed genetic and biochemical studies of the mechanisms guiding the generation of pyrroloketoindanes.

Single injection of PTH improves osteoclastic parameters of remodeling at a stress fracture site in rats.

Stress fractures (SFx) result from repetitive cyclical loading of bone. They are frequent athletic injuries and underlie atypical femoral fractures following long-term bisphosphonate (BP) therapy. We investigated the effect of a single PTH injection on the healing of SFx in the rat ulna. SFx was induced in 120 female Wistar rats (300 ± 15 g) during a single loading session. A single PTH (8 µg.100g-1 ) or vehicle (VEH) saline injection was administered 24 h after loading. Rats were divided into four groups (n = 15) and ulnae were examined 1, 2, 6, or 10 weeks following SFx. Two Toluidine Blue and TRAP-stained sections of the SFx were examined for histomorphometric analysis using Osteomeasure™ software. An increase in osteoclast number (N.Oc) and perimeter (Oc.Pm) was observed two weeks following PTH treatment (p < 0.01). At 6 weeks, bone formation was the main activity in BMUs. At 10 weeks, the proportion of healing along the SFx line remained 50% greater in PTH groups (p = 0.839), leading to a 43% reduction in the porosity area of BMU (p = 0.703). The main effect of time was a significant variable along the entire SFx remodeling cycle, with significant interactions between time and treatment type affecting (N.Oc) (p = 0.047) and (Oc.Pm) (p = 0.002). We conclude that a single PTH injection increases osteoclastogenesis by the second week of the remodeling cycle in a SFx in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Intramolecular cyclizations of polyketide biosynthesis: mining for a "Diels-Alderase"?

Despite the large number of naturally occurring metabolites existing for which enzymatic Diels-Alder reactions have been proposed as a key biosynthetic step, the actual number of enzymes thus far identified for these transformations is incredibly low. Even for those few enzymes identified, there is currently little biochemical or mechanistic evidence to support the label of a "Diels-Alderase." For several families of polyketide metabolites, the transformation in question introduces a rigid, cross-linked scaffold, leaving the remaining peripheral modifications and polyketide processing to provide the variation among the related metabolites. A detailed understanding of these modifications--how they are introduced and the tolerance of enzymes involved for alternate substrates--will strengthen biosynthetic engineering efforts toward related designer metabolites. This review addresses intramolecular cyclizations that appear to be consistent with enzymatic Diels-Alder transformations for which either the responsible enzyme has been identified or the respective biosynthetic gene cluster for the metabolite in question has been elucidated.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats.

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A2 (sPLA2) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA2-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA2-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA2 inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P<0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P<0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA2-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA2-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo.

Thiostrepton Variants Containing a Contracted Quinaldic Acid Macrocycle Result from Mutagenesis of the Second Residue.

The thiopeptides are a family of ribosomally synthesized and post-translationally modified peptide metabolites, and the vast majority of thiopeptides characterized to date possess one highly modified macrocycle. A few members, including thiostrepton A, harbor a second macrocycle that incorporates a quinaldic acid moiety and the four N-terminal residues of the peptide. The antibacterial properties of thiostrepton A are well established, and its recently discovered ability to inhibit the proteasome has additional implications for the development of antimalarial and anticancer therapeutics. We have conducted the saturation mutagenesis of Ala2 in the precursor peptide, TsrA, to examine which variants can be transformed into a mature thiostrepton analogue. Although the thiostrepton biosynthetic system is somewhat restrictive toward substitutions at the second residue, eight thiostrepton Ala2 analogues were isolated. The TsrA Ala2Ile and Ala2Val variants were largely channeled through an alternate processing pathway wherein the first residue of the core peptide, Ile1, is removed, and the resulting thiostrepton analogues bear quinaldic acid macrocycles abridged by one residue. This is the first report revealing that quinaldic acid loop size is amenable to alteration during the course of thiostrepton biosynthesis. Both the antibacterial and proteasome inhibitory properties of the thiostrepton Ala2 analogues were examined. While the identity of the residue at the second position of the core peptide influences thiostrepton biosynthesis, our report suggests it may not be crucial for antibacterial and proteasome inhibitory properties of the full-length variants. In contrast, the contracted quinaldic acid loop can, to differing degrees, affect both types of biological activity.

Saturation mutagenesis of TsrA Ala4 unveils a highly mutable residue of thiostrepton A.

Thiopeptides are post-translationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery's tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogues. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched hydrophobic residues are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogues were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

Osteocyte expression of caspase-3, COX-2, IL-6 and sclerostin are spatially and temporally associated following stress fracture initiation.

Stress fractures (SFxs) are debilitating injuries and exact mechanisms that initiate their repair incompletely understood. We hypothesised that osteocyte apoptosis and expression of cytokines and proteins such as sclerostin, VEGF, TGF-ß, COX-2 and IL-6 were early signalling events to facilitate the formation of periosteal woven bone and recruitment of osteoclast precursors to the site of remodelling. A SFx was created in the right ulna of mature female wistar rats using cyclic end loading. Rats were killed 1, 4 and 7 days after loading (n=5 per group). Standard histological staining was used to examine SFx morphology and immunohistochemistry to detect the localisation of these proteins and in situ hybridisation to detect mRNA along the SFx line or gene expression to quantify the target genes. Unloaded ulnae served as controls. The labelling index of caspase-3, COX-2 and IL-6 was significantly elevated in the region of SFxs at all time points compared with controls (P<0.001). In addition, the labelling index of sclerostin protein was significantly reduced in osteocytes adjacent to the SFx region when compared with controls at all three time points (P<0.001). Both VEGF and TGF-ß expressions were only localised in the woven bone. These data reinforce the involvement of osteocyte apoptosis in the healing of fatigue damage in bone, and demonstrate that local regulation of sclerostin, COX-2 and IL-6 are important signalling events associated with new bone formation and SFx remodelling.

Selective and non-selective cyclooxygenase inhibitors delay stress fracture healing in the rat ulna.

Anti-inflammatory drugs are widely used to manage pain associated with stress fractures (SFxs), but little is known about their effects on healing of those injuries. We hypothesized that selective and non-selective anti-inflammatory treatments would retard the healing of SFx in the rat ulna. SFxs were created by cyclic loading of the ulna in Wistar rats. Ulnae were harvested 2, 4 or 6 weeks following loading. Rats were treated with non-selective NSAID, ibuprofen (30 mg/kg/day); selective COX-2 inhibition, [5,5-dimethyl-3-3 (3 fluorophenyl)-4-(4 methylsulfonal) phenyl-2 (5H)-furanone] (DFU) (2.0 mg/kg/day); or the novel c5a anatagonist PMX53 (10 mg/kg/day, 4 and 6 weeks only); with appropriate vehicle as control. Quantitative histomorphometric measurements of SFx healing were undertaken. Treatment with the selective COX-2 inhibitor, DFU, reduced the area of resorption along the fracture line at 2 weeks, without affecting bone formation at later stages. Treatment with the non-selective, NSAID, ibuprofen decreased both bone resorption and bone formation so that there was significantly reduced length and area of remodeling and lamellar bone formation within the remodeling unit at 6 weeks after fracture. The C5a receptor antagonist PMX53 had no effect on SFx healing at 4 or 6 weeks after loading, suggesting that PMX53 would not delay SFx healing. Both selective COX-2 inhibitors and non-selective NSAIDs have the potential to compromise SFx healing, and should be used with caution when SFx is diagnosed or suspected.

In vivo production of thiopeptide variants.

Thiopeptides are a family of highly modified peptide metabolites, characterized by a macrocycle bearing a central piperidine/dehydropiperidine/pyridine ring, multiple thiazole rings, and several dehydrated amino acid residues. Thiopeptides have useful antibacterial, antimalarial, and anticancer properties but have not been adapted for human clinical applications, owing in part to their poor water solubility. In 2009, it was revealed that the thiopeptide scaffold is derived from a ribosomally synthesized precursor peptide subjected to extensive posttranslational modifications. Shortly thereafter, three groups developed two types of in vivo strategies to generate thiopeptide variants: precursor peptide mutagenesis and gene inactivation. The thiopeptide analogs and biosynthetic intermediates obtained from these studies provide much-needed insight into the biosynthetic process for these complicated metabolites. Furthermore, the in vivo production of variants can be employed to interrogate thiopeptide structure-activity relationships and may be useful to address the bioavailability issues plaguing these otherwise promising lead molecules. This chapter discusses the in vivo systems developed to generate thiopeptide variants.

Mutagenesis of the thiostrepton precursor peptide at Thr7 impacts both biosynthesis and function.

The seventh residue of thiostrepton is predicted to be critical for antibacterial activity. Substitution of Thr7 in the thiostrepton precursor peptide disrupts both biological activity and the successful biosynthesis of analogs.

Heterologous production of thiostrepton A and biosynthetic engineering of thiostrepton analogs.

Thiostrepton A 1, produced by Streptomyces laurentii ATCC 31255 (S. laurentii), is one of the more well-recognized thiopeptide metabolites. Thiostrepton A 1 and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens. Although numerous lines of evidence have established that the thiopeptides arise from the post-translational modification of ribosomally-synthesized peptides, few details have been revealed concerning this elaborate process. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton post-translational machinery. Due to the difficulties in the genetic manipulation of S. laurentii, the heterologous production of thiostrepton A 1 from an alternate streptomycete host was sought to facilitate the biosynthetic investigations of the peptide metabolite. The production of thiostrepton A 1 from the non-cognate hosts did not lend itself to be as robust as S. laurentii-based production, therefore an alternate strategy was pursued for the production of thiostrepton variants. The introduction of a fosmid used in the heterologous production of thiostrepton A 1, harboring the entire thiostrepton biosynthetic gene cluster, into the tsrA deletion mutant permitted restoration of thiostrepton A 1 production near to that of the wild-type level. The fosmid was then engineered to enable the replacement of wild-type tsrA. Introduction of expression fosmids encoding alternate TsrA sequences into the S. laurentii tsrA deletion mutant led to the production of thiostrepton variants retaining antibacterial activity, demonstrating the utility of this expression platform toward thiopeptide engineering.