Macrocyclization and Fatty Acid Modification during the Synthesis of Nonribosomal Peptides
Kopp, Florian
Nonribosomal peptides (NRPs) constitute a large and diverse class of pharmacologically important natural products that find useful therapeutic application as immunosuppressants, antibiotics, or anticancer agents. The biological activity of many of these compounds relies on the macrocyclic structure of their peptide backbone and the incorporation of a wide assortment of building blocks including proteinogenic and nonproteinogenic amino acids as well as modified fatty acid moieties. Particularly, these structural features are key determinants of nonribosomal lipopeptide antibiotics that are in the focus of this thesis.
To provide rapid access to these structurally demanding compounds, a chemoenzymatic approach towards the synthesis of the lipopeptide antibiotics daptomycin and A54145 was developed, based on the combined utilization of powerful solid phase peptide synthesis and the recombinant daptomycin and A54145 thioesterase (TE) domains. In vitro studies with these so-called peptide cyclases revealed their ability to catalyze the macrocyclization of linear peptidyl-thiophenol substrates with relaxed specificity for the cyclization nucleophile and electrophile. Ten lipopeptide variants were synthesized in order to explore the relatively sparse known acidic lipopeptide structure-activity relationship. Remarkably, this small library included a lipopeptide hybrid with a minimal inhibition concentration close to that of chemoenzymatic derived daptomycin as well as a bioactive macrolactam variant of A54145. Thus, single amino acid residues within the daptomycin and A54145 peptide sequences could be identified that are crucial for their antimicrobial potency.
Additionally, a unique and hitherto unknown type of imine macrocyclization as found for the cyanobacterial nostocyclopeptide (ncp) was investigated during the course of these studies. Experiments with ncp-CoA substrate mimics showed that a reductase (R) domain located at the C-terminal end of the ncp nonribosomal peptide synthetase (NRPS) is responsible for the reductive release of a reactive peptide aldehyde. Subsequently, imine macrocyclization occurs enzyme-independent under physiological pH conditions as proven with synthetic analogs of the ncp peptide aldehyde. An alanine scan experiment elucidated structural elements within the linear heptapeptide precursor that are essential for imine macrocyclization. Further, the biochemical characterization of ncp R also revealed its broad tolerance towards the C- and N-terminal amino acids of ncp substrate mimics.
In the third part of this work, the tailoring enzymes HxcO and HcmO from the calcium dependent antibiotic (CDA) trans 2,3 epoxyhexanoic acid biosynthetic pathway were chosen as a model system to investigate fatty acid modification during nonribosomal lipopeptide synthesis. While HxcO was characterized as a novel type of enzyme with dual function as an FAD-dependent fatty acid oxidase paired with intrinsic epoxidase activity, HcmO could be identified as a second epoxidase acting on 2,3-unsaturated fatty acids. Experiments with acyl-CoAs, acyl CoAs loaded onto an acyl carrier protein (ACP), and chemoenzymatically synthesized CDA variants revealed that both enzymes only accept ACP-bound substrates. To compare these ACP-bound HxcO and HcmO reaction products with synthetic standards a novel experimental approach had to be developed. Based on the thermodynamic activation inherent to thioester derivatives, the enzymatic products were cleaved from the ACP under mild conditions utilizing an amide ligation reaction and directly transformed into derivatives of smaller size suitable for HPLC-MS analysis. By the application of this versatile method the trans 2,3 epoxyhexanoic acid products of HxcO and HcmO were ascertained to have opposite absolute configuration, namely (2R,3S) and (2S,3R), respectively. In general, the established experimental approach holds great potential for the detailed analysis of all biochemical systems involving carrier protein-bound intermediates. These include integrated enzymes from NRPS and polyketide synthase (PKS) assembly lines or in trans acting tailoring enzymes
Philipps-Universität Marburg
Chemistry + allied sciences
https://doi.org/10.17192/z2008.0117
opus:1942
urn:nbn:de:hebis:04-z2008-01173
https://doi.org/10.17192/z2008.0117
Fatty Acid Modification
Peptidsynthetasen
Peptidsynthetasen
ppn:199044228
doctoralThesis
Philipps-Universität Marburg
Pepdtidmakrozyklisierung
Fachbereich Chemie
Makrozyklisierung und Fettsäuremodifikation während der Synthese nichtribosomaler Peptide
Fettsäuremodifikation
Peptidantibiotikum
opus:1942
English
Macrocyclization and Fatty Acid Modification during the Synthesis of Nonribosomal Peptides
Nichtribosomale Peptide (NRPs) bilden eine große und vielseitige Klasse von pharmakolo¬gisch bedeutsamen Naturstoffen, die als Immuno¬supressiva, Antibiotika oder Antikrebs-Wirkstoffe nützliche therapeutische Anwendung finden. Die biologische Aktivität vieler dieser Verbindungen beruht auf der makrozyklischen Struktur ihres Peptidrückgrats und der Inkorporierung eines großen Sortiments an Bausteinen, das proteinogene und nicht¬proteinogene Aminosäuren sowie modifizierte Fettsäurereste umfasst. Diese strukturellen Merkmale sind Schlüsseleigenschaften nichtribosomaler Lipopeptid¬antibiotika, die im Fokus dieser Arbeit stehen.
Um einen Zugang zu den strukturell anspruchsvollen Lipopeptiden Daptomycin und A54145 zu schaffen, wurde ein chemoenzymatischer Ansatz entwickelt, der auf dem kombinierten Einsatz von leistungsstarker Peptid-Festphasensynthese und rekombinanten Thioesterase Domänen basiert. In vitro Studien mit diesen so genannten Peptidzyklasen zeigten deren Fähigkeit, lineare Peptidyl-Thiophenol-Substrate mit entspannter Substratspezifität zu zyklisieren. Zehn Lipopeptidanaloga wurden hergestellt, um die relativ unbekannte Struktur-Aktivitäts Beziehung der aziden Lipopeptide zu erforschen. Bemerkenswerterweise enthielt diese kleine Bibliothek einen Lipopeptidhybrid mit einer minimalen Inhibitionskonzentration ähnlich der von chemoenzymatisch hergestelltem Daptomycin, sowie eine bioaktive A54145 Makrolactamvariante. Somit konnten einzelne Aminosäurereste in der Daptomycin- und A54145-Peptidsequenz identifiziert werden, die essentiell für deren antimikrobielle Eigenschaften sind.
Des Weiteren wurde im Rahmen dieser Arbeit eine bisher unbekannte Form der Iminmakrozyklisierung, wie sie für das cyanobakterielle Nostocyclopeptid (ncp) auftritt, untersucht. Experimente mit ncp-CoA-Substratmimikry zeigten, dass eine außerge¬wöhnliche Reduktase (R) Domäne, die sich am C-terminalen Ende der ncp nicht¬ribosomalen Peptidsynthetase (NRPS) befindet, für die reduktive Freisetzung eines reaktiven Peptidaldehyds verantwortlich ist. Anschließend verläuft die Iminmakro¬zyklisierung enzymunabhängig unter physiologischen pH Bedingungen, was durch synthetische ncp-Aldehyde belegt werden konnte. Ein Alanin-Scan Experiment identifizierte einzelne strukturelle Elemente im linearen Heptapeptidvorläufer, die ent-scheidend für den intramolekularen Zyklisierungsprozess sind.
Im dritten Teil dieser Arbeit wurden die CDA Tailoring Enzyme HxcO und HcmO des trans 2,3 Epoxyhexansäure Biosynthesewegs als Modellsystem gewählt, um die Fettsäuremodifikation in der Synthese nichtribosomaler Lipopeptide zu untersuchen. Während HxcO als ein neuer Enzymtyp mit dualer Funktion als FAD-abhängige Fettsäure Oxidase mit intrinsischer Epoxidaseaktivität charakterisiert wurde, konnte HcmO als eine zweite Epoxidase identifiziert werden, die an 2,3-ungesättigen Fettsäuren arbeitet. Experimente mit Acyl-CoAs, Acy-CoA beladenem Acyl Carrier Protein (ACP) sowie chemoenzymatisch hergestellten CDA Varianten ergaben, dass beide Enzyme nur ACP gebundene Substrate akzeptieren. Um diese ACP gebundenen Reaktionsprodukte mit synthetischen Standards vergleichen zu können, musste ein neuer experimenteller Ansatz entwickelt werden. Aufgrund der thermodynamischen Aktivierung von Thioesterderivaten, wurden die Enzymprodukte über eine Amidligationsreaktion unter milden Bedingungen vom ACP abgespalten und direkt in kleinere Derivate überführt, die für die HPLC MS-Analyse geeignet sind. Es wurde ermittelt, dass die trans 2,3 Epoxyhexansäure-Produkte von HxcO und HcmO entgegensetzte absolute Konfiguration, nämlich (2R,3S) bzw. (2S,3R), besitzen. Der etablierte experimentelle Ansatz birgt ein hohes Potenzial für die Analyse aller biochemischen Systeme, die an Carrier Protein-gebundenen Intermediaten arbeiten, wie z.B. integrierte Enzyme aus NRPSs und Polyketidsynthasen (PKSs) oder andere in trans agierende Tailoring Enzyme.
2011-08-10
https://archiv.ub.uni-marburg.de/diss/z2008/0117/cover.png
Chemistry + allied sciences
Chemie
urn:nbn:de:hebis:04-z2008-01173
Chemie
Nonribosomal Peptide Synthetases
129
application/pdf
ths
Prof. Dr.
Marahiel
M. A.
Marahiel, M. A. (Prof. Dr.)
Nonribosomal peptides (NRPs) constitute a large and diverse class of pharmacologically important natural products that find useful therapeutic application as immunosuppressants, antibiotics, or anticancer agents. The biological activity of many of these compounds relies on the macrocyclic structure of their peptide backbone and the incorporation of a wide assortment of building blocks including proteinogenic and nonproteinogenic amino acids as well as modified fatty acid moieties. Particularly, these structural features are key determinants of nonribosomal lipopeptide antibiotics that are in the focus of this thesis.
To provide rapid access to these structurally demanding compounds, a chemoenzymatic approach towards the synthesis of the lipopeptide antibiotics daptomycin and A54145 was developed, based on the combined utilization of powerful solid phase peptide synthesis and the recombinant daptomycin and A54145 thioesterase (TE) domains. In vitro studies with these so-called peptide cyclases revealed their ability to catalyze the macrocyclization of linear peptidyl-thiophenol substrates with relaxed specificity for the cyclization nucleophile and electrophile. Ten lipopeptide variants were synthesized in order to explore the relatively sparse known acidic lipopeptide structure-activity relationship. Remarkably, this small library included a lipopeptide hybrid with a minimal inhibition concentration close to that of chemoenzymatic derived daptomycin as well as a bioactive macrolactam variant of A54145. Thus, single amino acid residues within the daptomycin and A54145 peptide sequences could be identified that are crucial for their antimicrobial potency.
Additionally, a unique and hitherto unknown type of imine macrocyclization as found for the cyanobacterial nostocyclopeptide (ncp) was investigated during the course of these studies. Experiments with ncp-CoA substrate mimics showed that a reductase (R) domain located at the C-terminal end of the ncp nonribosomal peptide synthetase (NRPS) is responsible for the reductive release of a reactive peptide aldehyde. Subsequently, imine macrocyclization occurs enzyme-independent under physiological pH conditions as proven with synthetic analogs of the ncp peptide aldehyde. An alanine scan experiment elucidated structural elements within the linear heptapeptide precursor that are essential for imine macrocyclization. Further, the biochemical characterization of ncp R also revealed its broad tolerance towards the C- and N-terminal amino acids of ncp substrate mimics.
In the third part of this work, the tailoring enzymes HxcO and HcmO from the calcium dependent antibiotic (CDA) trans 2,3 epoxyhexanoic acid biosynthetic pathway were chosen as a model system to investigate fatty acid modification during nonribosomal lipopeptide synthesis. While HxcO was characterized as a novel type of enzyme with dual function as an FAD-dependent fatty acid oxidase paired with intrinsic epoxidase activity, HcmO could be identified as a second epoxidase acting on 2,3-unsaturated fatty acids. Experiments with acyl-CoAs, acyl CoAs loaded onto an acyl carrier protein (ACP), and chemoenzymatically synthesized CDA variants revealed that both enzymes only accept ACP-bound substrates. To compare these ACP-bound HxcO and HcmO reaction products with synthetic standards a novel experimental approach had to be developed. Based on the thermodynamic activation inherent to thioester derivatives, the enzymatic products were cleaved from the ACP under mild conditions utilizing an amide ligation reaction and directly transformed into derivatives of smaller size suitable for HPLC-MS analysis. By the application of this versatile method the trans 2,3 epoxyhexanoic acid products of HxcO and HcmO were ascertained to have opposite absolute configuration, namely (2R,3S) and (2S,3R), respectively. In general, the established experimental approach holds great potential for the detailed analysis of all biochemical systems involving carrier protein-bound intermediates. These include integrated enzymes from NRPS and polyketide synthase (PKS) assembly lines or in trans acting tailoring enzymes
2008
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
Lipopeptide Antbiotics
Kopp, Florian
Kopp
Florian
Peptidsynthese
monograph
Lipopeptidantibiotika
Peptidemacrocyclization
2008-03-17
2008-04-11
PRESERVATION_MASTER
VIEW
Image
PRESERVATION_MASTER