Dokument

Summary:
In syntrophischer Lebensweise oxidiert Syntrophus aciditrophicus Benzoat zu Acetat und CO2, während axenische Kulturen Crotonat zu Acetat und Cyclohexancarboxylat mit etwas Benzoat fermentieren. Genomische, proteomische und metabolische Analysen lassen vermuten, dass Abbau und Synthese von Benzoat mit Glutaconyl-CoA als zentralem Intermediat den gleichen Weg benutzen. In strikt anaeroben Bakterien wird Glutamat üblicherweise aus zwei Acetyl-CoA über Pyruvat, Oxalacetat, Citrat und 2-Oxoglutarat synthetisiert. Da im Genom von S. aciditrophicus kein Gen für Si-Citrat-Synthase gefunden wurde, vermuteten wir, dass Glutaconyl-CoA über 2-Hydroxyglutarat der Vorläufer von Glutamat sein könnte. Kürzlich wurde gezeigt, dass das rcs-Gen, das als Isopropylmalat/ Citramalat/ Homocitrat-Synthase annotiert ist, 49% Sequenzidentitäten mit dem Re-Citrat-Synthase-Gen aus Clostridium kluyveri aufweist. Wir haben deshalb das rcs-Gen kloniert und das rekombinante und mit einem C-terminalen Strep-tag versehene Protein in Escherichia coli überproduziert. Das Enzym wurde zur Homogenität gereinigt und mittels 14C-Markierung als Re-Citrat-Synthase charakterisiert. Die höchste spezifische Aktivität wurde mit Oxalacetat und Acetyl-CoA in Gegenwart von Co2+ erzielt. Pyruvat, 2-Oxoglutarat und 2-Oxoisovalerat konnten Oxalacetat nicht ersetzen; mit Propionyl-CoA war das Enzym ebenfalls inaktiv. Das reine Protein enthielt keine Metallionen; Co2+ oder auch Mn2+ waren nicht nur für die Aktivität notwendig sondern erhöhten auch die Stabilität. Obwohl Thiolreagenzien das Enzym partiell inaktivierten, scheint ein Cysteinrest nicht an der Katalyse beteiligt zu sein. Mit [2H3]Acetyl-CoA wurde ein geringer intermolekularer Isotopeneffekt (kH/kD = 1.4) gemessen. Vorläufige Versuche mit nativer Gelelektrophorese zeigen, dass das Enzym eine homodimere Struktur besitzt. Isotop-markierte Glutamate und Aspartate wurden aus S. aciditrophicus-Zellen isoliert, die axenisch auf unmarkiertem Crotonat mit [1-14C]Acetat oder 13CO2 gewachsen waren. Die Aminosäuren wurden entweder oxidativ decarboxyliert und über ihre Radioaktivität oder mittels 13C-NMR analysiert. Zusammen mit GC-MS-Daten der Universitäten Oklahoma und Washington, die [1-13C]Acetat benutzten, unterstützen unsere Ergebnisse eine Beteiligung der Re-Citrat-Synthase, obwohl eine unvollständige Äquilibrierung zwischen markiertem Acetat und ummarkiertem Crotonat in Betracht gezogen werden muss. Leider können die Wege über Re-Citrat-Synthase und Glutaconyl-CoA nicht allein durch Isotopenmarkierung unterschieden werden. Um die postulierte Reversibilität der Energie-konservierenden Glutaconyl-CoA-Decarboxylase (Gcd) zu untersuchen, insbesondere ob die Carboxylierung von Crotonyl-CoA durch einen elektrochemischen Na+-Gradienten getrieben wird, klonierten wir die im Genom von S. aciditrophicus vorhanden Gene gcdA, gcdB und gcdC. Die abgeleiteten Aminosäuresequenzen zeigen 52%, 51%, 46% und 42% Identitäten zu GcdA, B, C1 und C2 von Clostridium symbiosum, obwohl die (A+P) reiche Domäne von GcdC und gcdD fehlen. Die S. aciditrophicus-Gene wurden einzeln oder in den Kombinationen gcdAC und gcdABC in E. coli exprimiert. Nur GcdA, GcdC, und GcdAC konnten erfolgreich produziert werden. GcdA wurde als Carboxytransferase charakterisiert (2 mU/mg mit 5 mM Biotin als artifiziellem Akzeptor). Die Reinigung des Decarboxylase-Komplexes mittels Avidin-Affinitätschromatographie aus S. aciditrophicus-Zellen, die von einem Fermenter aus Leipzig stammten, war nicht erfolgreich. Zur Untersuchung der Mechanismus des durch Decarboxylierung getriebenen Na+-Transports, wären systematische Expressionsstudien von gcdB und eine Kristallisation des Komplexes erforderlich. Möglicherweise erleichtert das Fehlen der Aggregate verursachenden (A+P)-reichen Domäne von GcdC diese Aufgabe.

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