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 electron paramagnetic resonance (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 centre 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.

Unveiling Therapeutic Avenues for Crohn's Disease Management: Exploring Inhibitors for Adherent-Invasive Escherichia coli Propanediol Dehydratase.

INTRODUCTION: Inflammatory Bowel Disease (IBD) encompasses a group of chronic disorders distinguished by inflammation of the gastrointestinal tract. Among these, Crohn's Disease (CD) stands out as a complex and impactful condition due to challenges for both diagnosis and management, making it a cynosure of research. METHODS: In CD, there is the predominance of proinflammatory bacteria, including the Adherentinvasive Escherichia coli (AIEC) with virulence-associated metabolic enzyme Propanediol Dehydratase (pduC), which has been identified as a therapeutic target for the management of CD. Herein, molecular modeling techniques, including molecular docking, Molecular Mechanics with Generalized Born and Surface Area (MMGBSA), drug-likeness, and pharmacokinetics profiling, were utilized to probe the potentials of eighty antibacterial compounds to serve as inhibitors of pduC. RESULTS: The results of this study led to the identification of five compounds with promising potentials; the results of the molecular docking simulation revealed the compounds as possessing better binding affinities for the target compared to the standard drug (sulfasalazine), while Lipinski's rule of five-based assessment of their drug-likeness properties revealed them as potential oral drugs. MMGBSA free energy calculation and Molecular Dynamics (MD) simulation of the complexes formed a sequel to molecular docking, revealing the compounds as stable binders in the active site of the protein. CONCLUSION: Ultimately, the results of this study have revealed five compounds to possess the potential to serve as inhibitors of pduC of AIEC. However, experimental studies are still needed to validate the findings of this study.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

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.

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment.

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO2-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B12-dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B12-independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans-4-hydroxy-l-proline.

The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans-4-hydroxy-l-proline, the product of the most abundant human posttranslational modification. This "chemically guided functional profiling" workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities.

1,2-Propanediol Dehydration in Roseburia inulinivorans: STRUCTURAL BASIS FOR SUBSTRATE AND ENANTIOMER SELECTIVITY.

Glycyl radical enzymes (GREs) represent a diverse superfamily of enzymes that utilize a radical mechanism to catalyze difficult, but often essential, chemical reactions. In this work we present the first biochemical and structural data for a GRE-type diol dehydratase from the organism Roseburia inulinivorans (RiDD). Despite high sequence (48% identity) and structural similarity to the GRE-type glycerol dehydratase from Clostridium butyricum, we demonstrate that the RiDD is in fact a diol dehydratase. In addition, the RiDD will utilize both (S)-1,2-propanediol and (R)-1,2-propanediol as a substrate, with an observed preference for the S enantiomer. Based on the new structural information we developed and successfully tested a hypothesis that explains the functional differences we observe.

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 (α2ß2γ2) 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.

Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase.

Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eß31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R ß subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R ß and DD ß subunits, Gluß97 of DD would coordinate to Mg(2+). The complex of Eß97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluß97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.

[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.

Inactivation mechanism of glycerol dehydration by diol dehydratase from combined quantum mechanical/molecular mechanical calculations.

Inactivation of diol dehydratase during the glycerol dehydration reaction is studied on the basis of quantum mechanical/molecular mechanical calculations. Glycerol is not a chiral compound but contains a prochiral carbon atom. Once it is bound to the active site, the enzyme adopts two binding conformations. One is predominantly responsible for the product-forming reaction (G(R) conformation), and the other primarily contributes to inactivation (G(S) conformation). Reactant radical is converted into a product and byproduct in the product-forming reaction and inactivation, respectively. The OH group migrates from C2 to C1 in the product-forming reaction, whereas the transfer of a hydrogen from the 3-OH group of glycerol to C1 takes place during the inactivation. The activation barrier of the hydrogen transfer does not depend on the substrate-binding conformation. On the other hand, the activation barrier of OH group migration is sensitive to conformation and is 4.5 kcal/mol lower in the G(R) conformation than in the G(S) conformation. In the OH group migration, Glu170 plays a critical role in stabilizing the reactant radical in the G(S) conformation. Moreover, the hydrogen bonding interaction between Ser301 and the 3-OH group of glycerol lowers the activation barrier in G(R)-TS2. As a result, the difference in energy between the hydrogen transfer and the OH group migration is reduced in the G(S) conformation, which shows that the inactivation is favored in the G(S) conformation.

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).

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.

Catalytic roles of the metal ion in the substrate-binding site of coenzyme B12-dependent diol dehydratase.

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K(+) ion; however, QM/MM computations indicate that Ca(2+) ion is more reasonable as the metal ion because calculated Ca-O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K-O distances. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca(2+) ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K(+) ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca(2+) ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C-H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca(2+) ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335.

Diol dehydratase-reactivating factor is a reactivase--evidence for multiple turnovers and subunit swapping with diol dehydratase.

Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg(2+) by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg(2+). Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg(2+). Upon complex formation, one ß subunit was released from the (αß)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD ß subunit and the reactivase ß subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme: evidence that the substrate-coordinated metal ion is calcium.

The X-ray analyses of coenzyme B(12)-dependent diol dehydratase revealed two kinds of electron densities that correspond to metal ions in the active site. One is directly coordinated by substrate [Shibata, N., et al. (1999) Structure 7, 997-1008] and the other located near the adenine ring of the coenzyme adenosyl group [Masuda, J., et al. (2000) Structure 8, 775-788]. Both have been assigned as potassium ions, although the coordination distances of the former are slightly shorter than expected. We examined the possibility that the enzyme is a metalloenzyme. Apodiol dehydratase was strongly inhibited by incubation with EDTA and EGTA in the absence of substrate. The metal analysis revealed that the enzyme contains approximately 2 mol of tightly bound calcium per mole of enzyme. The calcium-deprived, EDTA-free apoenzyme was obtained by the EDTA treatment, followed by ultrafiltration. The activity of the calcium-deprived apoenzyme was dependent on Ca(2+) when assayed with 1 mM substrate. The K(m) for Ca(2+) evaluated in reconstitution experiments was 0.88 muM. These results indicate that the calcium is essential for catalysis. Ca(2+) showed a significant stabilizing effect on the calcium-deprived apoenzyme as well. It was thus concluded that the substrate-coordinated metal ion is not potassium but calcium. The potassium ion bound near the adenine ring would be the essential one for the diol dehydratase catalysis. Therefore, this enzyme can be considered to be a metal-activated metalloenzyme.

What is the identity of the metal ions in the active sites of coenzyme B12-dependent diol dehydratase? A computational mutation analysis.

What is the identity of the metal ion in the active sites of diol dehydratase? To address this question, we calculated the M-O bond lengths in the active sites using QM/MM calculations (M=K, Na, Mg, Ca). Our results show that the previous assignment of the metal ion in the substrate-binding site is wrong and that the identity of the metal ion is likely to be Ca2+. This is consistent with accumulated experimental evidence.

Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B12 -dependent glycerol and diol dehydratases.

Coenzyme B(12)-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the beta and gamma subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the beta and gamma subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the beta and gamma subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35-60) of the diol dehydratase beta subunit in addition to the diol dehydratase-specific extra-regions of beta and gamma subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.

Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis.

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Seralpha224, and of 6-NH(2), N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the epsilon-NH(2) group of Lysbeta135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Seralpha224 and Lysbeta135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Salpha224A, Salpha224N and Kbeta135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kbeta135A, Kbeta135Q and Kbeta135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the beta135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Salpha224A and Salpha224N mutants showed a k(cat)/k(inact) value that was less than 2% that of the wild-type, whereas for Lysbeta135 mutants this value was in the range 25-75%, except for the Kbeta135E mutant (7%). Unlike the wild-type holoenzyme, the Salpha224N and Salpha224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Seralpha224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Salpha224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5'-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.