Structural Characterization of the Heterobactin Siderophores from Rhodococcuserythropolis PR4 and Elucidation of their Biosynthetic Machinery
Zeyadi, Mustafa
Summary
The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior.
Philipps-Universität Marburg
Chemistry + allied sciences
urn:nbn:de:hebis:04-z2015-04168
opus:6475
https://doi.org/10.17192/z2015.0416
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Hydroxylation
Fachbereich Chemie
2015-12-23
urn:nbn:de:hebis:04-z2015-04168
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
https://archiv.ub.uni-marburg.de/diss/z2015/0416/cover.png
opus:6475
Chemistry + allied sciences
Chemie
English
Eisen
Strukturelle Charakterisierung des Heterobactin Siderophors aus Rhodococcuserythropolis PR4 und Untersuchung seiner biosynthetischen Maschinerie
Structural Characterization of the Heterobactin Siderophores from Rhodococcuserythropolis PR4 and Elucidation of their Biosynthetic Machinery
2015-12-10
application/pdf
Siderophore
Hydroxylierung
Rhodococcus
monograph
Chemie
doctoralThesis
Zeyadi, Mustafa
Zeyadi
Mustafa
Biosynthese
2015
Philipps-Universität Marburg
ths
Prof. Dr.
Marahiel
Mohamed A.
Marahiel, Mohamed A. (Prof. Dr.)
Rhodococcus
Biosynthesis
https://doi.org/10.17192/z2015.0416
Summary
The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior.
Zusammenfassung
Der Genus Rhodococcus gehört zu der Ordnung der Actinomycetales. Diese sind gram-positive Bakterien mit einem hohen GC-Gehalt, welche eine große Anzahl an Sekundärmetaboliten produzieren, die in der pharmazeutischen Industrie und in der Biotechnologie Anwendung gefunden haben. Die meisten dieser bioaktiven Verbindungen stammen entweder von nicht-ribosomalen Peptiden (NRP) oder Polyketiden (PK), von denen bisher jedoch nur wenige isoliert wurden. Nichtsdestotrotz wurde insbesondere innerhalb des Rhodococcus-Genus eine bemerkenswerte strukturelle Vielfalt von Siderophoren durch die Strukturaufklärung von Rhodochelin, Rhodobactin und Heterobactin A1 nachgewiesen. Deshalb wurde der Fokus dieser Arbeit auf die Isolierung und Charakterisierung von neuen Siderophor-Strukturen gelegt, um das chemische Potential in diesem Genus noch stärker hervorzuheben. In der vorliegenden Arbeit wird über die Isolierung, strukturelle Charakterisierung und Aufklärung des Biosyntheseursprungs des Siderophors Heterobactin berichtet. Dieser ist ein Catecholat-Hydroxamat-Mischtyp aus Rhodococcus erythropolis. Die Strukturaufklärung des extrahierten und gereinigten Siderophors Heterobactin A wurde mittels MSn-Analytik und NMR- Spektroskopie durchgeführt und zeigte die Anwesenheit einer besonderen Peptidbindung zwischen der Guanidingruppe eines Argininrestes und einer 2,3-Dihydroxybenzoatgruppe. Die beiden isolierten Heterobactin-Varianten S1 und S2 sind zudem Derivate von Heterobactin A, die eine Sulfonierung an den Aromaten aufweisen. Die bioinformatische Untersuchung des R. erythropolis PR4 Genoms und die anschließende biochemische Charakterisierung der putativen Biosynthesemaschinerie halfen das Gencluster zu identifizieren, das für die Biosynthese der Heterobactine verantwortlich ist. Interessanterweise verfügt die HtbG NRPS-Synthetase, die aus drei Modulen besteht, innerhalb ihres zweiten Moduls über eine nie zuvor beobachtete C-PCP-A Domänenorganisation, welche notwendig sein könnte für die korrekte Verlängerung der Peptidintermediate. Die vorliegende Arbeit korrigiert zudem die von Carrano et al. in 2001 beschriebene Heterobactin A Struktur. Die biochemische Charakterisierung der Monooxygenase HMO (kodiert vom Gen hmo) etablierte zudem die Biosyntheseroute der nicht-proteinogenen Aminosäure L-hOrn, bevor diese durch die NRPS-Maschinerie in das Peptidgerüst (Siderophor) eingefügt wird. Die Ergebnisse aus der strukturellen und biochemischen Charakterisierung der Heterobactine erlauben, zusammen mit der genetischen und biochemischen Charakterisierung der jeweiligen Biosynthesegencluster, den Vorschlag eines Biosynthesemodells für die Heterobactin-Assemblierung, In dieser arbeit wurde auch das Siderophor-Bindungsprotein htbH biochemisch untersucht, wobei seine hohe Affinität zum Mischtyp Catecholat-Hydroxamat Siderophor gezeigt werden konnte.
Siderophores
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