The coenzyme B 12 -dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E. coli strain producing both His 6 -tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably a 2 b 2 .The factor rapidly reactivated glycerol-or O 2 -inactivated hologlycerol dehydratase and activated the enzyme± cyanocobalamin complex in the presence of coenzyme B 12 , ATP, and Mg 21. The DhaF±DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF±DhaG complex of C. freundii efficiently crossactivated the enzyme±cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.Keywords: Citrobacter freundii; coenzyme B 12 ; glycerol dehydratase; glycerol fermentation; suicide inactivation.Glycerol dehydratase (EC 4.2.1.30) and diol dehydratase (EC 4.2.1.28) can each catalyze the conversion of glycerol, 1,2-propanediol, and 1,2-ethanediol to the corresponding aldehydes. These enzymatic reactions are known to proceed by a radical mechanism involving coenzyme B 12 as an essential cofactor. Both closely related enzymes have been extensively studied in genera of the Enterobacteriaceae such as Klebsiella and Citrobacter and in clostridia [1±7]. Glycerol and diol dehydratases are involved in the fermentation of glycerol and 1,2-propanediol, respectively [7±10]. It has been shown that these enzymes are similar in molecular masses and substrate spectra, but are different in monovalent cation selectivity patterns, immunochemical properties, affinity for coenzyme B 12 , and substrate specificity [11,12].The glycerol dehydratase of Citrobacter freundii is comprised of three different subunits (DhaBCE) and catalyzes the rate-limiting step in the anaerobic conversion of glycerol to 1,3-propanediol [4,9]. Glycerol dehydratases and diol dehydratases undergo irreversible inactivation by glycerol during catalysis [11,12]. Inactivation by glycerol involves irreversible cleavage of the Co±C bond of coenzyme B 12 , forming 5 H -deoxyadenosine and an alkylcobalamin-like species. Irreversible inactivation is then brought about by tight binding of the modified coenzyme [12]. Such suicide inactivation seems enigmatic, because glycerol is the gr...

Inhaltsverzeichnis II 2.4 Standardtechniken für das Arbeiten mit Nukleinsäuren 2.4.1 Behandlung von Geräten und Lösungen für das Arbeiten mit DNA 2.4.2 Puffer und Lösungen 2.4.3 Reinigung und Konzentrierung von DNA 2.4.3.1 Fällung von DNA 2.4.3.2 Mikrodialyse 2.4.3.3 Lithiumchloridfällung 2.4.3.4 Gelfiltration von DNA-Lösungen 2.4.3.5 Konzentrationsbestimmung von Nukleinsäuren 2.5 Isolierung von Nukleinsäuren 2.5.1 Isolierung von Gesamt-RNA aus C. freundii und Cl.