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Anal Biochem 150: 76-85 Miroux B, Walker JE (1996) Over-production of Proteins in Escherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. J Mol Biol 260: 289-298 https://doi.org/10.17192/z2011.0125 2011-08-11 https://archiv.ub.uni-marburg.de/diss/z2011/0125/cover.png Life sciences Biowissenschaften, Biologie Benzoat Syntrophus aciditrophicus thrives syntrophically on benzoate and axenically on crotonate, which is oxidized to acetate and reduced to cyclohexane carboxylate and some benzoate. Genomic, proteomic, and metabolic analyses suggested that degradation and synthesis of benzoate use the same pathway, whereby glutaconyl-CoA serves as central intermediate. In strictly anaerobic bacteria, glutamate is usually synthesized from two acetyl-CoA via pyruvate, oxaloacetate, citrate, and 2-oxoglutarate. As no gene for Si-citrate synthase has been detected in the genome of S. aciditrophicus, we speculated that glutaconyl-CoA via 2-hydroxyglutarate could be the precursor of 2-oxoglutarate for glutamate biosynthesis. Recently, the gene rcs, which is annotated as isopropylmalate/citratemalate/homocitrate synthase in S. aciditrophicus, has been shown to exhibit 49% sequence identity with that coding for Re-citrate from Clostridium kluyveri. We cloned rcs and overproduced the recombinant protein in Escherichia coli. The enzyme was purified aerobically and characterized biochemically as Re-citrate synthase. The highest achieved specific activity was 1.6 U/mg using oxaloacetate and acetyl-CoA as substrates in the presence of Co2+. Pyruvate, 2-oxoglutarate and 2-oxoisovalerate could not replace oxaloacetate; with propionyl-CoA also no activity was observed. No metal was detected in the recombinant protein. Besides Co2+ also Mn2+ stimulates the activity and stabilizes the enzyme. Sulfhydryl reagents partially inactivate the enzyme, but a cysteine residue seems not to be involved in the catalytic site. With [2H3]acetyl-CoA a low intermolecular deuterium isotope effect (kH/kD = 1.4) was measured. Preliminary native PAGE data indicate a homodimeric structure of the enzyme. Labeled glutamate and aspartate were extracted from S. aciditrophicus cells grown on unlabeled crotonate with [1-14C]acetate or 13CO2 and analyzed by oxidative decarboxylation and its radioactivity or by 13C-NMR, respectively. Together with GC-MS data from the universities of Oklahoma and Washington using [1-13C]acetate, the present results support the idea that Re-citrate synthase participates in glutamate biosynthesis, although an incomplete equilibration between labeled acetate and unlabeled crotonate must be considered. Unfortunately, the labeling pattern of glutamate derived from acetate via pyruvate, oxaloacetate and citrate cannot solely be distinguished from that via glutaconyl-CoA and 2-hydroxyglutarate. To study the proposed reversibility of the energy conserving glutaconyl-CoA decarboxylase (Gcd), especially whether the carboxylation of crotonyl-CoA is driven by an electrochemical Na+ gradient, we cloned the genes gcdA, gcdB, and gcdC detected in the genome of S. aciditrophicus. The deduced amino acid sequences show 52%, 51%, 46% and 42% identity to GcdA, B, C1 and C2 from Clostridium symbiosum, respectively, though the (A+P) rich domain of GcdC is missing and a gene for GcdD could not be detected. The S. aciditrophicus genes were expressed individually and in the combinations of gcdAC and gcdABC in E. coli, whereby only the productions of GcdA, GcdC, and GcdAC were successful. GcdA was characterized as carboxytranserase (2 mU/mg with 5 mM D-biotin as artificial acceptor). Purification of the decarboxylase complex by avidin affinity chromatography from S. aciditrophicus cells, grown in a fermenter in Leipzig, was not successful. To uncover the mechanism of transferring Na+ and CO2 in Gcd, a systematic approach of membrane protein overproduction and crystallization should be attempted. Perhaps the lack of the aggregate-forming (A+P) rich domain of GcdC facilitates crystallization. glutamate 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. Glutamat Fachbereich Biologie Biosynthese English ths Prof. Dr. Buckel Wolfgang Buckel, Wolfgang (Prof. Dr.) benzoate Erforschung der biosynthetischen Pfade von Glutamat und Benzoat bei Syntrophus aciditrophicus Syntrophus aciditrophicus Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg 2011-07-07 biosynthesis 2011 Exploring the biosynthetic pathways of glutamate and benzoate in Syntrophus aciditrophicus opus:3800 Syntrophus aciditrophicus