Analyse von Protease‐Inhibitor‐Interaktionen unter Nutzung evolvierbarer tricyclischer Microviridine

Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of compounds that combine great chemical complexity at low genetic costs. [1] The increasing knowledge about mechanisms and the scope of peptide-modifying enzymes has provided an avenue for the rational and random design of peptide libraries through synthetic biology methodologies. [2] Microviridins (1; see Scheme 1) are one of the most fascinating families of RiPPs with potent activities against various serine proteases. [3] Their unprecedented cage-like architecture results from the activity of two ATP-grasp ligases that introduce non-canonical lactone and lactam rings. [4] Full maturation of microviridins additionally requires the activity of a GNAT-type N-acetyltransferase. [4a] In vitro reconstitution studies have revealed a strict order of cyclization reactions and a stringent ring size requirement for microviridin with the large lactone ring being formed first, followed by the smaller lactone ring and the lactam ring. [4c] An increasing number of RiPPs was successfully reconstituted and diversified using chemo-enzymatic approaches that use chemically synthesized precursor peptides. [5] Expansion of the tool-kit for peptide modification has also enabled the design of "unnatural" peptide variants by modularizing different post-translational modification enzymes in vitro. [5] Microviridins feature a unique type of macrocyclization. [1,5] The full in vitro reconstitution of microviridins and thus their exploitation in chemo-enzymatic approaches was so far hampered by the long leader peptide that is essential for peptide maturation and the lack of an appropriate protease for leader peptide removal. Recent studies on lanthipeptide and cyanobactin biosynthetic enzymes have revealed that the functionality of the leader peptide does not necessarily require its direct linkage to the core peptide. [5a, 6] Leader peptides were shown to successfully activate the corresponding modifying enzymes when added in trans or were alternatively attached to the N-terminus of modifying enzymes. [5a...

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Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

Reyna‐González

Schmid

Petras

et al. 2016

Angewandte Chemie

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Eine sich selbst opfernde N‐Methyltransferase ist die Vorstufe des pilzlichen Sekundärmetaboliten Omphalotin

Ramm¹,

Krawczyk²,

Mühlenweg³

et al. 2017

Angewandte Chemie

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Die Forschung an ribosomal synthetisierten und posttranslational modifizierten Peptiden (RiPPs) hat das Verständnis von Biosynthesemechanismen zum Aufbau dieser Peptide weit vorangetrieben. Während die meisten Beispiele fürR iPPs von Bakterien stammen, gibt es außer den Amanitinen und Phalloidinen nur wenig bekannte Vertreter,d ie von Pilzen produziert werden. Das pilzlicheC yclopeptid Omphalotin At r ägt mehrere N-Methylierungen des Peptidrückgrats, eine Modifikation die so bisher nur fürn icht-ribosomal synthetisierte Peptide beschrieben wurde. Bei Untersuchungen des Genoms eines Omphalotin-produzierenden Pilzes konnten zwei Gene identifiziert werden, die an der Biosynthese beteiligt sind. Ein Gen kodiert fürd ie Methyltransferase OphMA, die eine Selbstmethylierung am eigenen C-Terminus durchführt und anschließend durcheine von einem zweiten Gen kodierte Prolyl-Oligopeptidase (OphP) erkannt, abgespalten und cyclisiert wird. Diese Arbeit zeigt einen neuen Biosynthesemechanismus fürR iPPs auf,b ei dem das Vorstufenpeptid Bestandteil des posttranslational modifizierenden Enzyms ist.Abbildung 3. Fragmentmassenspektrum des C-terminalen Peptids von OphMA mit annotierten b-und y-Ionen. Nicht exakt zuordenbare Methylierungen sind markiert (Me*).

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Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

et al. 2016

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Analyse von Protease‐Inhibitor‐Interaktionen unter Nutzung evolvierbarer tricyclischer Microviridine

Cited by 6 publications

References 20 publications

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

Eine sich selbst opfernde N‐Methyltransferase ist die Vorstufe des pilzlichen Sekundärmetaboliten Omphalotin

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

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