B12-dependent Diol Dehydratase Research Articles

The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in elimination of all the fluorides with formation of acetaldehyde.

3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and EPR spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical center compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.

Computational Study of Glycerol Binding within the Active Site of Coenzyme B12-Dependent Diol Dehydratase.

Molecular dynamics (MD) simulations have been employed for the first time to gain insight into the geometry of glycerol (GOL) bound within the active site of B12-dependent diol dehydratase (B12-dDDH). A peculiar feature of the B12-dDDH enzyme is that it undergoes suicidal inactivation by the substrate glycerol. To fully understand the inactivation mechanism, it is crucial to identify all possible interactions between GOL and the surrounding amino acid residues in the enzyme-substrate complex. Particularly important is the orientation of the C3-OH group in GOL since the presence of this OH group is the only difference between GOL and propanediol (PDO), a substrate for B12-dDDH that does not induce suicidal inactivation. The MD simulations indicate that glycerol can adopt two conformations that differ with respect to the orientation of the C3-OH group; in one conformer, the C3-OH group is oriented toward Ser301 (C3-OH···Ser301), and in the other toward Asp335 (C3-OH···Asp335). Although the former configuration is consistent with the crystal structure of B12-dDDH crystallized with cyanocobalamin (CNCbl) as the cofactor, MD simulations of this system suggest a substantial predominance of the latter conformer. A similar result with an even higher preference for the latter conformer is obtained for B12-dDDH with 5'-deoxyadenosylcobalamin (AdoCbl) as a cofactor. Employing QM/MM calculations it is found that the energy difference between the two conformers of GOL is very small in CNCbl B12-dDDH, where the slightly preferred conformer is C3-OH···Ser301. However, in AdoCbl B12-dDDH, this energy difference is higher, implying that GOL exists predominantly as the C3-OH···Asp335 conformer. These findings offer a new perspective for investigations of substrate-induced inactivation of the B12-dDDH enzyme.

Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum.

BackgroundProduction of the versatile bulk chemical 1,2-propanediol and the potential biofuel 1-propanol is still dependent on petroleum, but some approaches to establish bio-based production from renewable feed stocks and to avoid toxic intermediates have been described. The biotechnological workhorse Corynebacterium glutamicum has also been shown to be able to overproduce 1,2-propanediol by metabolic engineering. Additionally, C. glutamicum has previously been engineered for production of the biofuels ethanol and isobutanol but not for 1-propanol.ResultsIn this study, the improved production of 1,2-propanediol by C. glutamicum is presented. The product yield of a C. glutamicum strain expressing the heterologous genes gldA and mgsA from Escherichia coli that encode methylglyoxal synthase gene and glycerol dehydrogenase, respectively, was improved by additional expression of alcohol dehydrogenase gene yqhD from E. coli leading to a yield of 0.131 mol/mol glucose. Deletion of the endogenous genes hdpA and ldh encoding dihydroxyacetone phosphate phosphatase and lactate dehydrogenase, respectively, prevented formation of glycerol and lactate as by-products and improved the yield to 0.343 mol/mol glucose. To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol. Thus, B12-dependent diol dehydratase activity may be limiting 1-propanol production.ConclusionsProduction of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes. Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.

Computational Mutation Analysis of Enzymatic Reaction

Density functional theory (DFT) calculations are established as a useful research tool to investigate the structures and reactivity of biological systems; however, their high computational costs still restrict their applicability to systems of several tens up to a few hundred atoms. Recently, a combined quantum mechanical/molecular mechanical (QM/MM) approach has become an important method to study enzymatic reactions. In the past several years, we have investigated B12-dependent diol dehydratase using QM/MM calculations. The enzyme catalyzes chemically difficult reactions by utilizing the high reactivity of free radicals. In this paper, we explain our QM/MM calculations for the structure and reactivity of diol dehydratase and report key findings with respect to the catalytic roles of the active-site amino acid residues, computational mutational analysis of the active-site amino acid residues, assignment of the central metal ion, and function of the central metal ion. Our QM/MM calculations can correctly describe the structures and activation barriers of intermediate and transition states in the protein environment. Moreover, predicted relative activities of mutants are consistent with experimentally observed reactivity. These results will encourage the application of QM/MM research to the mechanistic study of enzymatic reactions, functional analysis of active-site residues, and rational design of enzymes with new catalytic functions.

Analysis of Enzymatic Reactions by Quantum Chemical Calculations

Density functional theory (DFT) is now extensively used as a research tool for the investigation of structure and reactivity of biological systems; however, its high computational demands still restrict the applicability of DFT to systems of a few tens up to one hundred atoms. A combined quantum mechanical/molecular mechanical (QM/MM) approach is applicable as an important method to study whole enzyme systems more than ten thousands atoms. We have investigated methane monooxygenase, dopamenie β-monooxygenase, tyrosinase, B12 dependent diol dehydratase, etc. using DFT and QM/MM calculations. In particular, we have done some computational mutation analyses about the amino acid residues at the active site of diol dehydratase. Our DFT and QM/MM calculations can correctly describe the structures and activation barriers of intermediates and transition states in the protein environment, and therefore, we successfully revealed the catalytic role of amino acid residues at the active site of diol dehydratase. Predicted relative activities of mutants are consistent with experimentally observed reaction rates. These results encourage us to apply QM/MM research to enzymatic reactions, functional analysis of active-site residues, and rational design of enzymes with new catalytic functions.

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.

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12‐dependent diol dehydratase and ethanolamine ammonia‐lyase

[Omega-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5', which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes.

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.

Structural Rationalization for the Lack of Stereospecificity in Coenzyme B12-dependent Diol Dehydratase

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca is apparently not stereospecific and catalyzes the conversion of both (R)- and (S)-1,2-propanediol to propionaldehyde. To explain this unusual property of the enzyme, we analyzed the crystal structures of diol dehydratase in complexes with cyanocobalamin and (R)- or (S)-1,2-propanediol. (R)- and (S)-isomers are bound in a symmetrical manner, although the hydrogen-bonding interactions between the substrate and the active-site residues are the same. From the position of the adenosyl radical in the modeled "distal" conformation, it is reasonable for the radical to abstract the pro-R and pro-S hydrogens from (R)- and (S)-isomers, respectively. The hydroxyl groups in the substrate radicals would migrates from C(2) to C(1) by a suprafacial shift, resulting in the stereochemical inversion at C(1). This causes 60 degrees clockwise and 70 degrees counterclockwise rotations of the C(1)-C(2) bond of the (R)- and (S)-isomers, respectively, if viewed from K+. A modeling study of 1,1-gem-diol intermediates indicated that new radical center C(2) becomes close to the methyl group of 5'-deoxyadenosine. Thus, the hydrogen back-abstraction (recombination) from 5'-deoxyadenosine by the product radical is structurally feasible. It was also predictable that the substitution of the migrating hydroxyl group by a hydrogen atom from 5'-deoxyadenosine takes place with the inversion of the configuration at C(2) of the substrate. Stereospecific dehydration of the 1,1-gem-diol intermediates can also be rationalized by assuming that Asp-alpha335 and Glu-alpha170 function as base catalysts in the dehydration of the (R)- and (S)-isomers, respectively. The structure-based mechanism and stereochemical courses of the reaction are proposed.

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.

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.

Intramolecular functionalisation by a methylene radical of a 1,2-diol and a vicinal amino alcohol: models for coenzyme B12-dependent diol dehydratase and ethanolamine ammonia lyase

Coenzyme B12-dependent diol dehydratase converts 1,2-diols (e.g. propane-1,2-diol) into the corresponding aldehyde and water. The similar enzyme ethanolamine ammonia lyase transforms vicinal aminoalcohols (e.g. 2-aminoethanol) into the corresponding aldehyde and ammonia. Model systems have been developed that replicate key features of the putative enzymatic mechanism, i.e. removal of a hydrogen atom from the CH2OH group of a 1,2-diol or vicinal aminoalcohol by a methylene radical derived from an alkylcobalt compound, and conversion of a 1,2-diol or vicinal aminoalcohol into a carbonyl compound and water or ammonia triggered by such a methylene radical. The models are based on alkyl(pyridine)bis(dimethylglyoximato)cobalt complexes [alkyl(pyridine)cobaloximes, Cbx], which were synthesised from appropriate organic precursors using standard methodology. The complexes contain a 1,2-diol [as in 4,5-dihydroxy-2,2-dimethylpentyl(pyridine)cobaloxime] or vicinal aminoalcohol [as in 6-amino-5-hydroxy-2,2-dimethylhexyl(pyridine)cobaloxime] tethered to the cobalt by a carbon chain. Photolysis or thermolysis of 4,5-dihydroxy-2,2-dimethylpentyl(pyridine)cobaloxime at pH 3 or 9 gave 4,4-dimethylpentanal. It is proposed that homolysis of the Co–C bond of 4,5-dihydroxy-2,2-dimethylpentyl(pyridine)cobaloxime induced by photolysis or thermolysis affords the 1,2-dihydroxy-4,4-dimethyl-1-pentyl radical via a 1,5-H shift, which is converted into the 4,4-dimethyl-1-oxo-2-pentyl radical, and hence 4,4-dimethylpentanal. The pathway for formation of the aldehyde was diagnosed using the specifically deuteriated analogue [5,5-2H2]-4,5-dihydroxy-2,2-dimethylpentyl(pyridine)cobaloxime, which gave [1,5-2H2]-4,4-dimethylpentanal accompanied by 3,3-dimethylbutanal on thermolysis or photolysis at pH 3. The protected model compound 2a was hydrolysed to 6-amino-5-hydroxy-2,2-dimethylhexyl(pyridine)cobaloxime, which was heated at pH 3 or 9 to give 5,5-dimethylhexan-2-one and ammonia.

Direct participation of potassium ion in the catalysis of coenzyme B(12)-dependent diol dehydratase.

The direct ion-dipolar interactions between potassium ion (K(+)) and the two hydroxyl groups of the substrate are the most striking feature of the crystal structure of coenzyme B(12)-dependent diol dehydratase. We carried out density-functional-theory computations to determine whether K(+) can assist the 1,2-shift of the hydroxyl group in the substrate-derived radical. Between a stepwise abstraction/recombination reaction proceeding via a direct hydroxide abstraction by K(+) and a concerted hydroxyl group migration assisted by K(+), only a transition state for the latter concerted mechanism was found from our computations. The barrier height for the transition state from the complexed radical decreases by only 2.3 kcal/mol upon coordination of the migrating hydroxyl group to K(+), which corresponds to a 42-fold rate acceleration at 37 degrees C. The net binding energy upon replacement of the K(+)-bound water for substrate was calculated to be 10.7 kcal/mol. It can be considered that such a large binding energy is at least partly used for the substrate-induced conformational changes in the enzyme that trigger the homolytic cleavage of the Co-C bond of the coenzyme and the subsequent catalysis by a radical mechanism. We propose here a new mechanism for diol dehydratase in which K(+) plays a direct role in the catalysis.

Toward a Consistent Mechanism for Diol Dehydratase Catalyzed Reactions: An Application of the Partial-Proton-Transfer Concept

Ab initio molecular orbital theory has been used to study the reactions catalyzed by the B12-dependent enzyme diol dehydratase. The calculations show that a pathway involving the 1,2-shift of a hydroxyl group is greatly facilitated by partial proton transfer to the migrating oxygen. These results suggest a conceptually simple mechanism for the rearrangement whose reaction rate is consistent with experiment. The inclusion of a gem-diol intermediate in the proposed pathway is in accordance with 18O-labeling experiments and thus overcomes important shortcomings in previously proposed mechanisms.

Coenzyme B12 dependent diol dehydratase model studies: chemical evidence against cobalt(B12) participation in the rearrangement step

Coenzyme B12 dependent diol dehydratase model studies: chemical evidence against cobalt(B12) participation in the rearrangement step

Model studies of coenzyme B12 dependent diol dehydratase. 1. Synthetic, physical property, and product studies of two key, cobalt-bound, putative diol dehydratase intermediates

Model studies of coenzyme B12 dependent diol dehydratase. 1. Synthetic, physical property, and product studies of two key, cobalt-bound, putative diol dehydratase intermediates

Model studies of coenzyme B12 dependent diol dehydratase. 2. A kinetic and mechanistic study focusing upon the cobalt participation or nonparticipation question

Model studies of coenzyme B12 dependent diol dehydratase. 2. A kinetic and mechanistic study focusing upon the cobalt participation or nonparticipation question

The mechanism of in sutu reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehydratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninylbutylcobalamin and adenosylethylcobalamin were essentially not displaceable by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Co beta ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are reactivated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism).