Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.     

Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium.

When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was kept under anaerobic conditions. n-Propanol and propionate were found in the medium as end products of this process at concentrations one-half that of 1,2-propanediol. As in Klebsiella pneumoniae (T. Toraya, S. Honda, and S. Fukui, J. Bacteriol. 139:39-47, 1979), a diol dehydratase which transforms 1,2-propanediol to propionaldehyde and the enzymes involved in a dismutation that converts propionaldehyde to n-propanol and propionate were induced in S. typhimurium cultures able to transform 1,2-propanediol anaerobically.     

The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.     

Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2

Salmonella enterica forms polyhedral organelles during coenzyme B(12)-dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B(12)-dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B(12)-dependent 1,2-PD degradation by S. enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B(12)-dependent diol dehydratase. Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB'CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB'JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO(2) fixation), suggesting that the S. enterica organelles and carboxysomes have a related multiprotein shell. In addition, S. enterica organelles contained four enzymes: B(12)-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the S. enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S. enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.     

The metabolism of 1,2-propanediol by the propylene oxide utilizing bacterium Nocardia A60

A bacterium designated Nocardia A60 was isolated for its capacity to utilize propylene oxide (1,2-epoxypropame) aerobically as a carbon and energy source for growth. Extracts of cells grown on the epoxide catalyzed the conversion of propylene oxide to 1,2-propanediol This epoxidase activity was absent in cells grown on 1,2-propanediol or succinate. During growth of the organism on propylene oxide and 1,2-propanediol it contained high levels of diol dehydratase (EC 4.2.1.28). Enhanced levels of propionyl-CoA carboxylase during growth on propylene oxide and 1,2-propanediol suggest that these compounds are metabolized via propionate and succinate.     

PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.     

Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae.

The presence of diol dehydratase and glycerol dehydratase was shown in several bacteria of Enterobacteriaceae grown anaerobically on 1,2-propanediol and on glycerol, respectively. Diol dehydratases of Enterobacteriaceae were immunologically similar, but distinct from that of Propionibacterium freudenreichii.     

Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724.

Immunochemical studies demonstrated that Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 produces only a single diol dehydratase whether grown on glycerol or on 1,2-propanediol. The enzyme was subject to induction by 1,2-diols and to catabolite repression reversed by cyclic AMP.     

The Propanediol Utilization (pdu) Operon of Salmonella enterica Serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.     

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ? Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol

The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.     

Energetic feasibility of hydrogen abstraction and recombination in coenzyme B(12)-dependent diol dehydratase reaction.

Coenzyme B(12) serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B(12)-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G* level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1,2-propanediol as substrate are 9.0 and 15.1 kcal/mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B(12)-dependent diol dehydratase reaction are energetically feasible.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

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 dehydratase-containing 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.     

Crystal structure of substrate free form of glycerol dehydratase

Glycerol dehydratase (GDH) and diol dehydratase (DDH) are highly homologous isofunctional enzymes that catalyze the elimination of water from glycerol and 1,2-propanediol (1,2-PD) to the corresponding aldehyde via a coenzyme B(12)-dependent radical mechanism. The crystal structure of substrate free form of GDH in complex with cobalamin and K(+) has been determined at 2.5 A resolution. Its overall fold and the subunit assembly closely resemble those of DDH. Comparison of this structure and the DDH structure, available only in substrate bound form, shows the expected change of the coordination of the essential K(+) from hexacoordinate to heptacoordinate with the displacement of a single coordinated water by the substrate diol. In addition, there appears to be an increase in the rigidity of the K(+) coordination (as measured by lower B values) upon the binding of the substrate. Structural analysis of the locations of conserved residues among various GDH and DDH sequences has aided in identification of residues potentially important for substrate preference or specificity of protein-protein interactions.     

Microbial metabolism of 1,2-propanediol studied by the Rumen Simulation Technique (Rusitec)

C zerkawski J.W., P iatkova M. & B reckenridge , G. 1984. Microbial metabolism of 1,2-propanediol studied by the Rumen. Simulation Technique (Rusitec). Journal of Applied Bacteriology , 56 , 81–94.
A series of experiments with the Rumen Simulation Technique (Rusitec) showed that 1,2-propanediol was metabolized efficiently by rumen micro-organisms and that the main end-products of fermentation were propionic and 2-methylbutyric acids. 'Propionaldehyde and n-propanol were also formed as intermediate compounds.' The effect of the diol on digestion of the basal diet appeared to be small with concentrate, or when the roughage was supplemented with additional nitrogen (urea). The decrease in the output of acetic and butyric acids was consistent with utilization of C₂ units for synthesis of 2-methylbutyric acid. The fermentation of 1,2-propanediol resulted in little or no increase in the output of additional microbial matter. The distribution of radioactivity from [1-¹⁴C] 1,2-propanediol confirmed that propionaldehyde and n -propanol were the primary products of metabolism of the diol and that the end-products were propionic and 2-methylbutyric acids, with very little labelling of microbial matter. Between 2% and 3% of radioactivity was found in gases and surprisingly the specific radioactivity of methane was higher than that of carbon dioxide, particularly during the initial stages of incubation. Possible pathways in the degradation of 1,2-propanediol by rumen micro-organisms are suggested and discussed in relation to similar reactions established in other systems.     

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.

The coenzyme B12-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 His6-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 alpha2beta2. The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. 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 cross-activated 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.     

Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions, and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.     

Fermentation of primary alcohols and diols and pure culture of syntrophically alcohol-oxidizing anaerobes

Ethanol, propanol, ethylene glycol, 1,2-propanediol, 1,2-butanediol, acetoin, diacetyl, and 2,3-pentanedione were used as substrates for enrichment and isolation of alcohol-oxidizing fermentative bacteria. Diacetyl and 2,3-pentanedione proved to be highly toxic. With the other substrates, various kinds of bacteria could be isolated which were assigned to three different metabolic groups: (i) homoacetogenic bacteria, and (ii) bacteria forming propionate as reduced end product were isolated from freshwater sources; (iii) bacteria disproportionating acetoin and 1,2-diols to acids and primary alcohols were isolated from marine sediments. The latter oxidized primary alcohols to fatty acids in the presence of hydrogen-oxidizing partners. Syntrophically ethanol-oxidizing cocultures enriched with primary alcohols could be separated with 1,2-diols as substrates into an alcohol-oxidizing organism and a hydrogen-oxidizing homoacetogen. The pathways of alcohol conversion in the disproportionating isolates were studied in detail. Growth experiments as well as enzymological studies demonstrated that acetoin and 1,2-diols were degraded via acetaldehyde which was also an intermediate in syntrophic oxidation of primary alcohols. The environmental importance of the various metabolic types isolated was assessed by most-probable-number enumerations.     

Resolution of the coenzyme B-12-dependent dehydratases of Klebsiella sp. and Citrobacter freundii.

Diol dehydratase (1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) are shown to be distinct, separable enzymes that occur individually or together in different strains of Klebsiella sp. Anaerobic growth with propan-1,2-diol induces diol dehydratase alone, whereas glycerol fermentation induces both enzymes in K. pneumoniae ATCC 25955 and in Citrobacter freundii NCIB 3735. The dehydratases can be resolved by polyacrylamide-gel electrophoresis or separated by anion-exchange chromatography alone. Sucrose density gradient centrifugation failed to distinguish the enzymes and indicated a molecular weight of 1.9 . 10(5) for both. The enzymes can be assayed individually, even when present in the same crude extract, using the 67-fold difference in their Km values for coenzyme B-12. For both enzymes inactivation kinetics are observed with glycerol as substrated, and monovalent cations influence both the inactivation rate and catalytic rate of the reaction.