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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...

To isolate genes encoding coenzyme B 12 -dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratasecontaining microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C 2 to C 4 ) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive proteinencoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.

The N'-methyltetrahydomethanopterin (H,MPT) :coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyltetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei GO1 by a two-step solubilization with s-octyl-P-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 pM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei GO1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na+/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methylcobalamin could substitute for methyl-H,MPT.