The Siderophore-Interacting Protein YqjH Acts as a Ferric Reductase in Different Iron Assimilation Pathways of <i>Escherichia coli</i>

Siderophore-interacting proteins (SIPs), such as YqjH from Escherichia coli, are widespread among bacteria and commonly associated with iron-dependent induction and siderophore utilization. In this study, we show by detailed biochemical and genetic analyses the reaction mechanism by which the YqjH protein is able to catalyze the release of iron from a variety of iron chelators, including ferric triscatecholates and ferric dicitrate, displaying the highest efficiency for the hydrolyzed ferric enterobactin complex ferric (2,3-dihydroxybenzoylserine)3. Site-directed mutagenesis revealed that residues K55 and R130 of YqjH are crucial for both substrate binding and reductase activity. The NADPH-dependent iron reduction was found to proceed via single-electron transfer in a double-displacement-type reaction through formation of a transient flavosemiquinone. The capacity to reduce substrates with extremely negative redox potentials, though at low catalytic rates, was studied by displacing the native FAD cofactor with 5-deaza-5-carba-FAD, which is restricted to a two-electron transfer. In the presence of the reconstituted noncatalytic protein, the ferric enterobactin midpoint potential increased remarkably and partially overlapped with the effective E1 redox range. Concurrently, the observed molar ratios of generated Fe­(II) versus NADPH were found to be ∼1.5-fold higher for hydrolyzed ferric triscatecholates and ferric dicitrate than for ferric enterobactin. Further, combination of a chromosomal yqjH deletion with entC single- and entC fes double-deletion backgrounds showed the impact of yqjH on growth during supplementation with ferric siderophore substrates. Thus, YqjH enhances siderophore utilization in different iron acquisition pathways, including assimilation of low-potential ferric substrates that are not reduced by common cellular cofactors

Loading Peptidyl-Coenzyme A onto Peptidyl Carrier Proteins: A Novel Approach in Characterizing Macrocyclization by Thioesterase Domains

Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates. To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase. A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP. With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates

A Four-Enzyme Pathway for 3,5-Dihydroxy-4-methylanthranilic Acid Formation and Incorporation into the Antitumor Antibiotic Sibiromycin

The antitumor antibiotic sibiromycin belongs to the class of pyrrolo[1,4]benzodiazepines (PBDs) that are produced by a variety of actinomycetes. PBDs are sequence-specific DNA-alkylating agents and possess significant antitumor properties. Among them, sibiromycin, one of two identified glycosylated PBDs, displays the highest DNA binding affinity and the most potent antitumor activity. In this study, we report the elucidation of the precise reaction sequence leading to the formation and activation of the 3,5-dihydroxy-4-methylanthranilic acid building block found in sibiromycin, starting from the known metabolite 3-hydroxykynurenine (3HK). The investigated pathway consists of four enzymes, which were biochemically characterized in vitro. Starting from 3HK, the SAM-dependent methyltransferase SibL converts the substrate to its 4-methyl derivative, followed by hydrolysis through the action of the PLP-dependent kynureninase SibQ, leading to 3-hydroxy-4-methylanthranilic acid (3H4MAA) formation. Subsequently the NRPS didomain SibE activates 3H4MAA and tethers it to its thiolation domain, where it is hydroxylated at the C5 position by the FAD/NADH-dependent hydroxylase SibG yielding the fully substituted anthranilate moiety found in sibiromycin. These insights about sibiromycin biosynthesis and the substrate specificities of the biosynthetic enzymes involved may guide future attempts to engineer the PBD biosynthetic machinery and help in the production of PBD derivatives

Harnessing the Chemical Activation Inherent to Carrier Protein-Bound Thioesters for the Characterization of Lipopeptide Fatty Acid Tailoring Enzymes

Here, we report a new experimental approach utilizing an amide ligation reaction for the characterization of acyl carrier protein (ACP)-bound reaction intermediates, which are otherwise difficult to analyze by traditional biochemical methods. To explore fatty acid tailoring enzymes of the calcium-dependent antibiotic (CDA) biosynthetic pathway, this strategy enabled the transformation of modified fatty acids, covalently bound as thioesters to an ACP, into amide ligation products that can be directly analyzed and compared to synthetic standards by HPLC-MS. The driving force of the amide formation is the thermodynamic activation inherent to thioester-bound compounds. Using this novel method, we were able to characterize the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of CDA, revealing a new type of FAD-dependent oxidase HxcO with intrinsic enoyl-ACP epoxidase activity, as well as a second enoyl-ACP epoxidase, HcmO. In general, our approach should be widely applicable for the in vitro characterization of other biosynthetic systems acting on carrier proteins, such as integrated enzymes from NRPS and PKS assembly lines or tailoring enzymes of fatty and amino acid precursor synthesis

Peptide Macrocyclization: The Reductase of the Nostocyclopeptide Synthetase Triggers the Self-Assembly of a Macrocyclic Imine

Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought

Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in <i>Bacillus subtilis</i>

<div><p>Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in <i>Bacillus subtilis</i> and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme <i>b</i> biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the <i>in vitro</i>-generated heme <i>b</i> co-factor upon Fra-mediated iron transfer was confirmed by using the <i>B</i>. <i>subtilis</i> nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme <i>b</i> co-factor, hence representing a key player in the iron-dependent physiology of <i>B</i>. <i>subtilis</i>.</p></div

Consecutive Enzymatic Modification of Ornithine Generates the Hydroxamate Moieties of the Siderophore Erythrochelin

Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-l-ornithine (l-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of l-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of l-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-l-ornithine (l-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating l-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of l-haOrn was accomplished in coupled assays employing both the l-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-l-ornithine starting from l-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd

Chemoenzymatic Design of Acidic Lipopeptide Hybrids: New Insights into the Structure−Activity Relationship of Daptomycin and A54145^†

The acidic lipopeptides, including the clinically approved antibiotic daptomycin, constitute a class of structurally related branched cyclic peptidolactones and peptidolactams synthesized by nonribosomal peptide synthetases (NRPSs). In this study, the excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, which were chemically produced by solid phase peptide synthesis. Applying this chemoenzymatic strategy, we generated derivatives of A54145 and daptomycin as well as hybrid molecules of both compounds. Bioactivity determination of the derived cyclic molecules revealed new insights into the structure−activity relationship of the acidic lipopeptide family. The general importance of several amino acid positions, including two conserved aspartic acid residues, was confirmed to be substantial for antibiotic potency. As a robust macrocyclization catalyst, the peptide cyclase excised from A54145 synthetase is the first cyclase of a branched cyclic lipopeptide, which catalyzes both macrolactonization and macrolactamization. The results presented herein illustrate the advantages of combining organic synthesis with natural product biosynthetic enzymes to explore the interplay between structural features and biological activity

Formylation Domain: An Essential Modifying Enzyme for the Nonribosomal Biosynthesis of Linear Gramicidin

Formylation is an important part of ribosomal peptide synthesis of prokaryotes. In nonribosomal peptide synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin nonribosomal peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl−valine for the initiation of the gramicidin biosynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the dipeptide formyl−valyl−glycine was found when assayed in the presence of either formyl donor. The proven formylation activity of the first domain of LgrA represents a novel tailoring enzyme in nonribosomal peptide synthesis

Overview of the proposed iron uptake and distribution pathways in <i>B</i>. <i>subtilis</i>.

<p>Iron is taken up either in the form of ferric siderophore complexes by transporters specific for endogenous or exogenous ferric siderophores [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref030" target="_blank">30</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref031" target="_blank">31</a>], or in form of non-siderophore bound iron [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref032" target="_blank">32</a>]. Iron in the cytosol is released from the siderophore complexes either in the ferric or ferrous state [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref030" target="_blank">30</a>], and may undergo complete reduction until it is transferred to the iron chaperone frataxin (Fra) for intracellular trafficking to iron co-factor biogenesis systems, including iron-sulphur cluster (Fe-S) assembly [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref011" target="_blank">11</a>] or, as characterised in this study, heme assembly. Further possible interactions with iron sensors/regulators or iron storage components have not yet been addressed yet.</p