The production of siderophore analogues using precursor-directed biosynthesis.

Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.

Measurement of lead, mercury, and cadmium in blood donors in Canada.

BACKGROUND: Fetal and neonatal exposure to lead is associated with irreversible adverse effects on neural development. There is no reliable threshold for lead effect, so limiting exposure is recommended. A significant correlation has been reported between post-transfusion blood lead level (BLL) in infants and lead levels in transfused RBC units. We measured levels of lead, mercury, and cadmium, in Canadian donor blood to investigate if concerning levels for neonatal transfusion exist. STUDY DESIGN AND METHODS: Whole blood samples from blood donors (n = 2529) were shipped cold within 7 days of donation. All permanent blood donation clinics across Canada were sampled. Twelve of these permanent clinics and 8 mobile clinics with a greater potential for having higher lead or mercury levels were oversampled. Heavy metals were measured by inductively coupled plasma mass spectrometry. RESULTS: Of all donations, 2.2% (lead) and 0.4% (mercury) had levels higher than the recommended thresholds for safe neonatal transfusion. BLLs were higher in males but there was no significant difference in the blood mercury levels of males versus females. Cadmium levels were higher in females. There was a positive correlation between donor age and levels of heavy metals, with lead having the strongest correlation (r = 0.47, p < .0001). Three clinics in close proximity to two lead-producing mines were among the clinics with the highest BLLs. Significantly higher blood mercury levels were observed in coastal clinics. CONCLUSION: Our data on donor blood heavy metal levels supports considering blood transfusion as an exposure source to heavy metals and encourages informed selection of blood units for transfusion to vulnerable groups.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster under iron limitation.

IMPORTANCE: Campylobacter jejuni is a bacterium that is prevalent in the ceca of farmed poultry such as chickens. Consumption of ill-prepared poultry is thus the most common route by which C. jejuni infects the human gut to cause a typically self-limiting but severe gastrointestinal illness that can be fatal to very young, old, or immunocompromised people. The lack of a vaccine and an increasing resistance to current antibiotics highlight a need to better understand the mechanisms that make C. jejuni a successful human pathogen. This study focused on the functional components of one such mechanism-a molecular system that helps C. jejuni thrive despite the restriction on growth-available iron by the human body, which typically defends against pathogens. In providing a deeper understanding of how this system functions, this study contributes toward the goal of reducing the enormous global socioeconomic burden caused by C. jejuni.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster in iron scavenging.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be antecedent to highly debilitating long-term sequelae. Expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution by each of the downstream genes ( fetABCDEF ) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA , fetB, fetC , and fetD , suggesting a role in iron acquisition for each encoded protein. FetA expression was modulated by iron-availability but not dependent on the presence of FetB, FetC, FetD, FetE or FetF. Functions of the putative thioredoxins FetE and FetF were redundant in iron scavenging, requiring a double deletion (Δ fetEF ) to exhibit a growth defect. C. jejuni FetE was expressed and the structure solved to 1.50 Å, revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than E. coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo . This study advances our understanding of the contributions by the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge towards mitigating C. jejuni -related morbidity and mortality.

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins.

Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.

Exploring hydroxamic acid inhibitors of HDAC1 and HDAC2 using small molecule tools and molecular or homology modelling.

Hydroxamic acid compounds 1-10 containing a N-hydroxycinnamamide scaffold and a 4-(benzylamino)methyl cap group that was either unsubstituted (1) or substituted with one (2-4) or two (5-10) methoxy groups in variable positions were prepared as inhibitors of Zn(II)-containing histone deacetylases (HDACs). The 3,4- (9) and 3,5- (10) bis-methoxy-substituted compounds were the least potent against HeLa nuclear extract, HDAC1 and HDAC2. Molecular modelling showed methoxy groups in the 3-, 4- and 5-position, but not the 2-position, had unfavourable steric interactions with the G32-H33-P34 triad on a loop at the surface of the HDAC2 active site cavity. An HDAC1 homology model showed potential ionic (E243..K288) and cation-pi (K247..F292) interactions between helix 10 and helix 11 that were absent in HDAC2 ((G243..K288) and (K247..V292)). This surface-located interhelical constraint could inform the design of bitopic HDAC1 and HDAC2 selective ligands using an allosteric approach, and/or protein-protein interaction (PPI) inhibitors.

Analogues of desferrioxamine B (DFOB) with new properties and new functions generated using precursor-directed biosynthesis.

Desferrioxamine B (DFOB) is a siderophore native to Streptomyces pilosus biosynthesised by the DesABCD enzyme cluster as a high affinity Fe(III) chelator. Although DFOB has a long clinical history for the treatment of chronic iron overload, limitations encourage the development of new analogues. This review describes a recent body of work that has used precursor-directed biosynthesis (PDB) to access new DFOB analogues. PDB exploits the native biosynthetic machinery of a producing organism in culture medium augmented with non-native substrates that compete against native substrates during metabolite assembly. The method allows access to analogues of natural products using benign methods, compared to multistep organic synthesis. The disadvantages of PDB are the production of metabolites in low yield and the need to purify complex mixtures. Streptomyces pilosus medium was supplemented with different types of non-native diamine substrates to compete against native 1,5-diaminopentane to generate DFOB analogues containing alkene bonds, fluorine atoms, ether or thioether functional groups, or a disulfide bond. All analogues retained function as Fe(III) chelators and have properties that could broaden the utility of DFOB. These PDB studies have also added knowledge to the understanding of DFOB biosynthesis.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Advances in the Chemical Biology of Desferrioxamine B.

Desferrioxamine B (DFOB) was discovered in the late 1950s as a hydroxamic acid metabolite of the soil bacterium Streptomyces pilosus. The exquisite affinity of DFOB for Fe(III) identified its potential for removing excess iron from patients with transfusion-dependent hemoglobin disorders. Many studies have used semisynthetic chemistry to produce DFOB adducts with new properties and broad-ranging functions. More recent approaches in chemical biology have revealed some nuances of DFOB biosynthesis and discovered new DFOB-derived drugs and radiometal imaging agents. The current and potential applications of DFOB continue to inspire a rich body of chemical biology research focused on this bacterial metabolite.

Dimeric and trimeric homo- and heteroleptic hydroxamic acid macrocycles formed using mixed-ligand Fe(III)-based metal-templated synthesis.

Macrocyclic hydroxamic acids coordinate Fe(III) with high affinity as part of siderophore-mediated bacterial iron acquisition. Trimeric hydroxamic acid macrocycles, such as desferrioxamine E (DFOE), are prevalent in nature, with fewer dimeric macrocycles identified, including putrebactin (pbH2), avaroferrin (avH2), bisucaberin (bsH2) and alcaligin (alH2). This work used metal-templated synthesis (MTS) to pre-assemble complexes between one equivalent of Fe(III) and two equivalents of 4-((4-aminobutyl)(hydroxy)amino)-4-oxobutanoic acid (BBH) or 4-((5-aminopentyl)(hydroxy)amino)-4-oxobutanoic acid (PBH). Following peptide coupling, the respective Fe(III) complexes of pbH2 or bsH2 were formed, which analysed by LC-MS under acidic pH as [Fe(pb)]+ ([M]+, m/zobs 426.1) or [Fe(bs)]+ ([M]+, m/zobs 454.2). The mixed-ligand 1:1:1 Fe(III):BBH:PBH system furnished [Fe(pb)]+ and [Fe(bs)]+, together with chimeric [Fe(av)]+ ([M]+, m/zobs 440.2). The deviation from the expected 1:2:1 distribution of [Fe(pb)]+:[Fe(av)]+:[Fe(bs)]+ to 1:3.2:1.6 suggested the MTS-mediated formation of dimeric macrocycles could be influenced by steric effects in the pre-complex and/or cavity size, as governed by the monomer. 21-Membered avH2 defined the lower boundary of the optimal architecture. Mixed-ligand MTS between Fe(III):PBH-d4:ret-PBH at 1:1.5:1.5, where ret-PBH=3-(6-amino-N-hydroxyhexanamido)propanoic acid, gave four Fe(III)-loaded trimeric hydroxamic acid macrocycles in a distribution of 1.0:3.0:2.9:1.1 that closely matched the expected distribution 1:3:3:1 for a system without any kinetic and/or thermodynamic bias. Apo-macrocycles pbH2, avH2 and bsH2 were produced upon incubation with diethylenetriaminepentaacetic acid (DTPA) and co-eluted with a biosynthetic mixture of the native macrocycles. The work has demonstrated the utility of single- and mixed-ligand MTS for producing a variety of homo- and heteroleptic dimeric hydroxamic acid macrocycles as Fe(III) complexes and free ligands.

Exploiting the biosynthetic machinery of Streptomyces pilosus to engineer a water-soluble zirconium(iv) chelator.

The water solubility of a natural product-inspired octadentate hydroxamic acid chelator designed to coordinate Zr(iv)-89 has been improved by using a combined microbiological-chemical approach to engineer four ether oxygen atoms into the main-chain region of a methylene-containing analogue. First, an analogue of the trimeric hydroxamic acid desferrioxamine B (DFOB) that contained three main-chain ether oxygen atoms (DFOB-O3) was generated from cultures of the native DFOB-producer Streptomyces pilosus supplemented with oxybis(ethanamine) (OBEA), which competed against the native 1,5-diaminopentane (DP) substrate during DFOB assembly. This precursor-directed biosynthesis (PDB) approach generated a suite of DFOB analogues containing one (DFOB-O1), two (DFOB-O2) or three (DFOB-O3) ether oxygen atoms, with the latter produced as the major species. Log P measurements showed DFOB-O3 was about 45 times more water soluble than DFOB. Second, a peptide coupling chain-extension reaction between DFOB-O3 and the synthetic ether-containing endo-hydroxamic acid monomer 4-((2-(2-aminoethoxy)ethyl)(hydroxy)amino)-4-oxobutanoic acid (PBH-O1) gave the water soluble tetrameric hydroxamic acid DFOB-O3-PBH-O1 as an isostere of sparingly water soluble DFOB-PBH. The complex between DFOB-O3-PBH-O1 and natZr(iv), examined as a surrogate measure of the radiolabelling procedure, analysed by LC-MS as the protonated adduct ([M + H]+, m/zobs = 855.2; m/zcalc = 855.3), with supporting HRMS data. The use of a microbiological system to generate a water-soluble analogue of a natural product for downstream semi-synthetic chemistry is an attractive pathway for developing new drugs and imaging agents. The improved water solubility of DFOB-O3-PBH-O1 could facilitate the synthesis and purification of downstream products, as part of the ongoing development of ligands optimised for Zr(iv)-89 immunological PET imaging.

Reverse Biosynthesis: Generating Combinatorial Pools of Drug Leads from Enzyme-Mediated Fragmentation of Natural Products.

A combinatorial pool of hydroxamic acid fragments as potential metalloprotein drug leads was generated from the enzymatic hydrolysis of the natural product desferrioxamine B (DFOB). DFOB is a metabolite produced by Streptomyces pilosus for iron acquisition, and can be selectively catabolised by Niveispirillum irakense to access carbon for growth. The supernatant of a DFOB-supplemented culture of N. irakense was analysed by LC-MS at intervals over 168 h. This identified a mixture of endo-hydroxamic acid fragments that contained reactive terminal groups. The supernatants from two cultures (at 48 h and 168 h) were reacted with 1,8-naphthalic anhydride in a microwave synthesiser to generate pools of scriptaid analogues, which were screened against ZnII -containing histone deacetylases (HDACs) and FeIII -containing 5-lipoxygenase (5-LO). Compound S2 showed relative potency against 5-LO (IC50 =59 µm; BWA4C, 17 µm); it was 28-fold more selective towards 5-LO than HDAC1. Compound S1 inhibited HDAC1 but not 5-LO. Enzyme-mediated reverse biosynthesis could yield new benefits from structurally complex natural products in drug design.