Dokument

Zusammenfassung:
Zusammen mit der Pyruvat-Formiat-Lyase, der anaeroben Ribonukleotid-Reduktase, der Co-Enzym B12-unabhängigen Diol-Dehydratase, der Cholintrimethylamin-Lyase und der 4-Hydroxyphenylacetat-Decarboxylase gehört die (R)-Benzylsuccinat-Synthase zur Familie der Glycylradikalenzyme. Die (R)-Benzylsuccinat-Synthase (BSS) katalysiert die Initiierung des anaeroben Abbaus von Toluol mit einer Addition der Methylgruppe des Toluols an die Doppelbindung von Fumarat unter Bildung von (R)-Benzylsuccinat als Produkt der Reaktion. Sie besteht aus drei Untereinheiten mit 98, 8,5 und 6,5 kDa, die ein Heterohexamer α2β2γ2 mit einer Gesamtmasse von 220 kDa bilden. Die großen α-Untereinheiten enthalten jeweils einen essentiellen Glycin- und Cysteinrest, die in allen Glycylradikal-Enzymen konserviert sind. Das aktive Zentrum des Enzyms befindet sich in der großen α-Untereinheit, wobei die beiden kleinen Untereinheiten jeweils ein Fe4S4-Cluster unbekannter Funktion beinhalten. Die β-Untereinheit könnte aufgrund ihrer Lage in der kürzlich aufgeklärten Struktur den Zugang des Substrats an das katalytische Zentrum der α-Untereinheit regulieren. An ihrem konservierten Glycinrest am C-Terminus der großen Untereinheit wird sie posttranslational durch das SAM-abhängige aktivierende Enzym BssD aktiviert, wobei ein freies Radikal an einem konservierten Glycin-Rest eingeführt wird und ein Glycylradikal entsteht. Die Strukturgene bssC, A und B sind mit dem Gen für das aktivierende Enzym bssD und weiteren Genen unbekannter Funktion in einem Toluol-induzierbaren Operon organisiert. Neben Toluol werden andere methylierte Kohlenwasserstoffe wie n-Alkane, Cycloalkane, p-Cresol, 2-Methylnaphthalin, p-Cymol (in Thauera sp. pCyN2) und Ethylbenzol durch die Anlagerung von Fumarat für den weiteren Abbauweg zu Acyl-CoA-Thioestern aktiviert. Die katalysierenden Enzyme werden als Fumarat-addierende Enzyme (FAEs) zusammengefasst. Als katalytischer Mechanismus der Benzylsuccinat-Synthase-Reaktion wurde die Bildung eines enzymgebundenen Substratradikals aus Toluol vorgeschlagen, das anschließend mit Fumarat zu einem Produktradikal reagiert. Durch die Re-Abstrahierung eines Wasserstoffatoms vom katalytischen Cysteinrest Cys493 entsteht im nächsten Schritt das Produktradikal. In diesem Projekt sollten weitere Informationen über den Reaktionsmechanismus der BSS mittels biochemischer und spektroskopischer Analysen erhalten werden. Bereits in der Vergangenheit wurden durch EPR-Analysen organische Radikale als Reaktionsintermediate identifiziert, die aus verschiedenen Substratanaloga hervorgegangen sind. In dieser Arbeit wurden die EPR-spektroskopischen Analysen mit Benzylalkohol als potenten Inhibitor der BSS, die von Markus Hilberg begonnen wurden, fortgesetzt. Dabei wurden Fragen, die in der Abhandlung von Hilberg (2013) noch offen geblieben sind, beantwortet. Die eindeutige Identifizierung der neuen organischen Radikalspezies, die für die Aufklärung des Mechanismus der BSS dienlich ist, steht noch aus. Weiterhin ist es zum ersten Mal gelungen, in vivo aktivierte BSS heterolog bei Inkubation mit Benzoat als Substrat überzuproduzieren und BSS-Aktivität im Rohextrakt nachzuweisen. Es sollte die Rolle wichtiger Aminosäuren, die das katalytische Zentrum der BSS bilden, in Bezug auf die Substratspezifität, die Enantiospezifität und den generellen Katalysemechanismus des Enzyms aufgeklärt werden. Es wurden mehrere Aminosäuren in der Nähe des katalytischen Zentrums der α-Untereinheit der BSS mutiert, um das entsprechende Enzym in Aromatoleum aromaticum Stamm EbN1 SR7 überzuproduzieren. Mit den erhaltenen Enzymaktivitäten der Extrakte sollten die Enzymaktivitäten und die Nutzung verschiedener Substrate jeweils im Vergleich zum Wildtyp-Enzym analysiert werden. Um gänzlich auszuschließen, dass eine gemessene Aktivität auf das Wildtypenzym zurückzuführen ist und um die Rolle der unbekannten Gene bssEFGH aufzuklären, wurde das bss-Operon vollständig (bssDCABEFGH) und partiell (bssEFGH) erfolgreich deletiert. Durch Verwendung von chiralem radioaktiv markiertem (R)-und (S)-Toluol mit je einem der drei verschiedenen Wasserstoffisotope an der Methylgruppe ist es in dieser Arbeit gelungen, die Stereospezifität der Benzylsuccinat-Synthase aufzuklären. Bei einer Reaktion dieser Substrate mit Fumarat kommt es an der Methylgruppe des Toluols zu einer Inversion der Konfiguration.

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