Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis.

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974.

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.

Differential Biosynthesis and Roles of Two Ferrichrome-Type Siderophores, ASP2397/AS2488053 and Ferricrocin, in Acremonium persicinum.

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum- and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.

Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase.

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.

Nonclinical Development of Next-generation Site-specific HER2-targeting Antibody-drug Conjugate (ARX788) for Breast Cancer Treatment.

Conventional antibody-drug conjugates (ADC) utilize native surface-exposed lysines or cysteines on the antibody of interest to conjugate cytotoxic payload. The nonspecific conjugation results in a mixture with variable drug-to-antibody ratios (DAR), conjugation sites, and ADCs that are often unstable in systemic circulation. ARX788 is an ADC consisting of a HER2-targeting antibody site-specifically conjugated with a potent antitubulin cytotoxic drug-linker, AS269. The site-specific conjugation is achieved by first incorporating the nonnatural amino acid, para-acetyl phenylalanine (pAF), into the antibody, followed by covalent conjugation of AS269 to the pAF to form a highly stable oxime bond resulting in a DAR 2 ADC. ARX788 exhibits significant, dose-dependent antitumor activity against HER2- expressing breast and gastric xenograft tumors. Pharmacokinetic (PK) studies in multiple species showed the highly stable nature of ARX788 with overlapping PK profiles for the intact ADC and total antibody. Metabolism studies demonstrated that pAF-AS269 was the sole major metabolite of ARX788, with no evidence for the release of free drug often observed in conventional ADCs and responsible for adverse side effects. Furthermore, ARX788 demonstrated a favorable safety profile in monkeys with a highest nonseverely toxic dose of 10 mg/kg, which was well above the efficacious dose level observed in preclinical tumor models, thus supporting clinical development of ARX788.

Construction of sulfur-containing moieties in the total synthesis of natural products.

Covering: January 2013 to September 2018Sulfur-containing natural products are a large class of significant functional molecules. Many of these compounds exhibit potent biological activities and pharmacological properties; in fact, some of them have been developed into important drugs. The total synthesis of sulfur-containing natural products is a subject that has long attracted significant attention from synthetic organic chemists; to achieve this goal, various methods have been developed over the past years. This review surveys total syntheses of sulfur-containing natural products that introduce sulfur atoms using different sulfurization agents to construct related sulfur-containing moieties.

Identification of the Enzymes Mediating the Maturation of the Seryl-tRNA Synthetase Inhibitor SB-217452 during the Biosynthesis of Albomycins.

Albomycin δ2 is a sulfur-containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l-serine-thioheptose dipeptide partial structure, known as SB-217452, has been found to be the active seryl-tRNA synthetase inhibitor component of albomycin δ2 . Herein, it is demonstrated that AbmF catalyzes condensation between the 6'-amino-4'-thionucleoside with the d-ribo configuration and seryl-adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3'-epimerization to produce SB-217452 with the d-xylo configuration, which is catalyzed by the radical S-adenosyl-l-methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB-217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB-217452.

The effects of structurally different siderophores on the organelles of Pinus sylvestris root cells.

MAIN CONCLUSION: Siderophores are a driver of Pinus sylvestris root responses to metabolites secreted by pathogenic and mycorrhizal fungi. Structurally different siderophores regulate the uptake of Fe by microorganisms and may play a key role in the colonization of plants by beneficial or pathogenic fungi. Siderophore action, however, may be dependent on the distribution of Fe within cells. Here, the involvement of siderophores in determining the changes of organelle morphology and element composition of some cellular fractions of root cells in Pinus sylvestris to trophically diverse fungi was investigated. Changes in the morphology and concentrations of different elements within organelles of root cells in response to three structurally different siderophores were examined by transmission electron microscopy combined with energy-dispersive X-ray spectroscopy. Weak development of mitochondrial cristae and the deposition of backup materials in plastids occurred in the absence of Fe in the structures of triacetylfusarinine C and ferricrocin. In response to metabolites of both pathogenic and mycorrhizal fungi, Fe accumulated mainly in the cell walls and cytoplasm. Fe counts increased in all of the analyzed organelles in response to applications of ferricrocin and triacetylfusarinine C. Chelation of Fe within the structure of siderophores prevents the binding of exogenous Fe, decreasing the abundance of Fe in the cell wall and cytoplasm. The concentrations of N, P, K, Ca, Mn, Cu, Mg, and Zn also increased in cells after applications of ferricrocin and triacetylfusarinine C, while the levels of these elements decreased in the cell wall and cytoplasm when Fe was present within the structure of the siderophores. These results provide insight into the siderophore-driven response of plants to various symbionts.

Biosynthetic Origin of the Atypical Stereochemistry in the Thioheptose Core of Albomycin Nucleoside Antibiotics.

Albomycins are peptidyl thionucleoside natural products that display antimicrobial activity against clinically important pathogens. Their structures are characterized by a thioheptose with atypical stereochemistry including a d-xylofuranose ring modified with a d-amino acid moiety. Herein it is demonstrated that AbmH is a pyridoxal 5'-phosphate (PLP)-dependent transaldolase that catalyzes a threo-selective aldol-type reaction to generate the thioheptose core with a d-ribofuranose ring and an l-amino acid moiety. The conversion of l-to d-amino acid configuration is catalyzed by the PLP-dependent epimerase AbmD. The d- ribo to d- xylo conversion of the thiofuranose ring appears according to gene deletion experiments to be mediated by AbmJ, which is annotated as a radical S-adenosyl-l-methionine (SAM) enzyme. These studies establish several key steps in the assembly of the thioheptose core during the biosynthesis of albomycins.

Total synthesis and antimicrobial evaluation of natural albomycins against clinical pathogens.

Development of effective antimicrobial agents continues to be a great challenge, particularly due to the increasing resistance of superbugs and frequent hospital breakouts. There is an urgent need for more potent and safer antibiotics with novel scaffolds. As historically many commercial drugs were derived from natural products, discovery of antimicrobial agents from complex natural product structures still holds a great promise. Herein, we report the total synthesis of natural albomycins δ1 (1a), δ2 (1b), and ε (1c), which validates the structures of these peptidylnucleoside compounds and allows for synthetic access to bioactive albomycin analogs. The efficient synthesis of albomycins enables extensive evaluations of these natural products against model bacteria and clinical pathogens. Albomycin δ2 has the potential to be developed into an antibacterial drug to treat Streptococcus pneumoniae and Staphylococcus aureus infections.

Ferrichrome Has Found Its Match: Biomimetic Analogues with Diversified Activity Map Discrete Microbial Targets.

Siderophores provide an established platform for studying molecular recognition principles in biological systems. Herein, the preparation of ferrichrome (FC) biomimetic analogues varying in length and polarity of the amino acid chain separating between the tripodal scaffold and the pendent FeIII chelating hydroxamic acid groups was reported. Spectroscopic and potentiometric titrations determined their iron affinity to be within the range of efficient chelators. Microbial growth promotion and iron uptake studies were conducted on E. coli, P. putida and U. maydis. A wide range of siderophore activity was observed in the current series: from a rare case of a species-specific growth promotor in P. putida to an analogue matching FC in cross-phylum activity and uptake pathway. A fluorescent conjugate of the broad-range analogue visualized siderophore destination in bacteria (periplasmic space) vs. fungi (cytosol) mapping new therapeutic targets. Quantum dots (QDs) decorated with the most potent FC analogue provided a tool for immobilization of FC-recognizing bacteria. Bacterial clusters formed around QDs may provide a platform for their selection and concentration.

Ferrichrome identified from Lactobacillus casei ATCC334 induces apoptosis through its iron-binding site in gastric cancer cells.

Ferrichrome is known to be a siderophore, but it was recently identified as a tumor-suppressive molecule derived from Lactobacillus casei ATCC334 ( L. casei). In the present study, we investigated the effects of ferrichrome in gastric cancer cells. Cell lines and xenograft models treated with ferrichrome demonstrated growth suppression. The expression levels of cleaved poly (adenosine diphosphate-ribose) polymerase, and cleaved caspase-9 were increased by ferrichrome treatment. Although the tumor-suppressive effects of ferrichrome were almost completely diminished by the iron chelation, the reduction in the intracellular iron by ferrichrome did not correlate with its tumor-suppressive effects. An exhaustive docking simulation indicated that iron-free ferrichrome can make stable conformations with various mammalian molecules, including transporters and receptors. In conclusion, probiotic-derived ferrichrome induced apoptosis in gastric cancer cells. The iron binding site of ferrichrome is the structure responsible for its tumor suppressive function.

Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae.

The pathogenic Streptococcus pneumoniae (S. pneumoniae) has evolved a special mechanism such as pneumococcal iron acquisition ATP binding cassette (PiaABC) to take up siderophore-iron from its host. The cell-surface lipoprotein PiaA, a key component of PiaABC, is the primary receptor to bind ferrichrome (Fc). To study the structure-function relationship of PiaA, three conservative amino-acid residues, Trp63, Trp158 and Phe255, in the hydrophobic barrel of the metal binding site of PiaA, were individually and collectively mutated to alanine; and the resulted single-point mutants, W63A, W158A and F255A, and triple mutant W63A/W158A/F255A were characterized by using biochemical and biophysical methods. Experiments showed that wild-type PiaA (WT-PiaA) and the single-point mutant proteins bound Fc with a similar kinetics mode, but the reaction rate of W158A was lower than that for WT-PiaA. The binding affinity of W158A toward Fc was significantly weaker than that of the WT-PiaA-Fc (wild-type PiaA bound with Fc) interaction. Furthermore, the absence of Trp158 in the protein led to a significant impact on the secondary structure of PiaA, resulting in a labile conformational structure of W158A, with impaired resistance to thermal and chemical denaturation. Collectively, Trp158 is a crucial residue for binding Fc, playing an important role in stabilizing the PiaA-Fc complex. This study revealed the critical role of the conserved tryptophan residues in Fc-binding protein PiaA, and provided valuable information for understanding the Fc transport mechanism mediated by PiaA or its homologous proteins in bacteria.

Crystal structure of FhuD at 1.6†Šresolution: a ferrichrome-binding protein from the animal and human pathogen Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistant S. pseudintermedius strains are emerging, resembling the global health threat of S. aureus. Therefore, it is increasingly important to characterize potential targets for intervention against S. pseudintermedius. Here, FhuD, an S. pseudintermedius surface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15 °C. The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at 1.6 Å resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target of S. pseudintermedius.

Biomimetic ferrichrome: structural motifs for switching between narrow- and broad-spectrum activities in P. putida and E. coli.

A series of novel ferrichrome (FC) analogs was designed based on the X-ray structure of FC in the FhuA transporter of Escherichia coli. Two strategies were employed: the first strategy optimized the overall size and relative orientation of H-bonding interactions. The second strategy increased H-bonding interactions by introducing external H-donors onto analogs' backbone. Tris-amino templates were coupled to succinic or aspartic acid, and the second carboxyl was used for hydroxamate construction. Succinic acid provided analogs without substituents, whereas aspartic acid generated analogs with external amines (i.e. H-donors). All analogs had similar physicochemical properties, yet the biological activity in Pseudomonas putida and E. coli showed great variation. While some analogs targeted specifically P. putida, others were active in both strains thus exhibiting broad-spectrum activity (as in native FC). Narrow-spectrum or species-specificity might find application in microbial diagnostic kits, while broad-spectrum recognition may have advantages in therapeutics as siderophore-drug conjugates. The differences in the structure and range of microbial recognition helped us in formulating guidelines for minimal essential parameters required for inducing broad-spectrum activity.

Tenellin acts as an iron chelator to prevent iron-generated reactive oxygen species toxicity in the entomopathogenic fungus Beauveria bassiana.

Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress.

Identification of the hydroxamate siderophore ferricrocin in Cladosporium cladosporioides.

The hydroxamate siderophore ferricrocin was identified in Cladosporium cladosporioides growth medium by solid phase extraction and ultra high pressure liquid chromatography coupled to a time of flight mass spectrometer (UHPLC/QTOF-MS). Both desferricrocin and ferricrocin were detected in the extracellular medium assisted by high resolution mass spectrometry. This is the first identification of a hydroxamate siderophore in Cladosporium cladosporioides. This finding emphasizes the common meaning of ferricrocin in fungi.

FhuA interactions in a detergent-free nanodisc environment.

TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study ß-barrel membrane proteins in a membrane-like environment.

Lipoprotein FtsB in Streptococcus pyogenes binds ferrichrome in two steps with residues Tyr137 and Trp204 as critical ligands.

Lipoprotein FtsB is a component of the FtsABCD transporter that is responsible for ferrichrome binding and uptake in the Gram-positive pathogen Streptococcus pyogenes. In the present study, FtsB was cloned and purified from the bacteria and its Fch binding characteristics were investigated in detail by using various biophysical and biochemical methods. Based on the crystal structures of homogeneous proteins, FtsB was simulated to have bi-lobal structure forming a deep cleft with four residues in the cleft as potential ligands for Fch binding. With the assistance of site-directed mutagenesis, residue Trp204 was confirmed as a key ligand and Tyr137 was identified to be another essential residue for Fch binding. Kinetics experiments demonstrated that Fch binding in FtsB occurred in two steps, corresponding to the bindings to Tyr137 at N-lobe and Trp204 from C-lobe, respectively, and so that closing the protein conformation. Without either residue Tyr137 or Trp204, Fch binding in the protein as mutants Fch-Y137A and Fch-W204A may have a loose conformation, resembling the apo-proteins in proteolysis resistance and migration behaviors in native gel. This study revealed the inconsistence in the key amino acids among Fch-binding proteins from Gram-positive and -negative bacteria, providing interesting findings for understanding the differences between Gram-positive and -negative bacteria in the mechanism of iron uptake via siderophore (Fch) binding and transport.

Isolation and characterisation of a ferrirhodin synthetase gene from the sugarcane pathogen Fusarium sacchari.

FSN1, a gene isolated from the sugar-cane pathogen Fusarium sacchari, encodes a 4707-residue nonribosomal peptide synthetase consisting of three complete adenylation, thiolation and condensation modules followed by two additional thiolation and condensation domain repeats. This structure is similar to that of ferricrocin synthetase, which makes a siderophore that is involved in intracellular iron storage in other filamentous fungi. Heterologous expression of FSN1 in Aspergillus oryzae resulted in the accumulation of a secreted metabolite that was identified as ferrirhodin. This siderophore was found to be present in both mycelium and culture filtrates of F. sacchari, whereas ferricrocin is found only in the mycelium, thus suggesting that ferricrocin is an intracellular storage siderophore in F. sacchari, whereas ferrirhodin is used for iron acquisition. To our knowledge, this is the first report to characterise a ferrirhodin synthetase gene functionally.