Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B₁₂‐dependent glycerol dehydratase of Citrobacter freundii

Abstract

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...

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

“…All of the glycerol and diol dehydratases characterized so far belong to class II of coenzyme B12-containing enzymes (50,51). The radical mechanism of action of these enzymes, involving coenzyme B 12 as essential cofactor, was initially proposed by Abeles and coworkers (52,53).…”

MeaB Is a Component of the Methylmalonyl-CoA Mutase Complex Required for Protection of the Enzyme from Inactivation

“…Evidence That MeaB Is Not a Reactivating Factor of MCMOne possibility for the function of MeaB could be to facilitate release of cofactor from the enzyme, in analogy to the reactivase of other B 12 -dependent enzymes (6,26). It is possible that after the catalytic cycle, cofactor (intact or inactivated) cannot be released to be regenerated and MeaB might function in this regard.…”

“…Other B 12 -dependent enzymes (glycerol and diol dehydratase) undergo suicide inactivation by the substrate during catalysis (6,26,27). The inactivation involves irreversible cleavage of the Co-C bond of AdoCbl, forming 5Ј-deoxyadenosine and an alkylcobalamin-like species (27).…”

Microbial Conversion of Glycerol to 1,3-Propanediol by an Engineered Strain of Escherichia coli

“…Furthermore, 3-HPA levels above 30 mM have been shown to inhibit glycerol dehydratase (1). Recent studies also have shown that glycerol dehydratases undergo irreversible suicide inactivation by glycerol (9,17,31), despite glycerol being the substrate and the dehydratase being essential for glycerol breakdown. Finally, one of the key limitations of the application of this biological process to industry is that it requires the coenzyme B 12 -dependent glycerol dehydratase, necessitating the addition of large amounts of a high-cost molecule, vitamin B 12 , to the culture medium.…”