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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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The methods of homologous high-level expression and simple large-scale purification for coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer α6β6. Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B12, the binding of 1 mol of the inhibitor per mol of the αβ unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme. [ABSTRACT FROM PUBLISHER]

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, Mg2+ 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). H2S 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. [ABSTRACT FROM PUBLISHER]

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (kcat/klinact) of 630 for ethanolamine ammonialyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the kcat/kinact value for the imidazolyl-cobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase. [ABSTRACT FROM AUTHOR]

Coenzyme B12 serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B12-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 B12-dependent diol dehydratase reaction are energetically feasible [ABSTRACT FROM AUTHOR]

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 B12-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 step wise 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°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. [ABSTRACT FROM AUTHOR]

EPR spectra were measured upon incubation of the complex of diol dehydratase with coenzyme analogs in the presence of 1, 2-propanediol, a physiological substrate. When the analog in which the D-ribose moiety of the nucleotide loop was replaced by a trimethylene group was used as coenzyme, essentially the same EPR spectrum as that with adenosylcobalamin was obtained. The higher-field doublet and the lower-field broad signals derived from an organic radical and low-spin Co(II) of cob(II)alamin, respectively, were observed. With the imidazolyl counterpart, base-on cob(II)alamin-like species accumulated, but signals due to an organic radical quickly disappeared. When a coenzyme analog lacking the nucleotide moiety was incubated with apoenzyme in the presence of substrate, the EPR spectrum resembling cob(II)inamide was obtained, but no signals due to an organic radical were observed. From these results, it was concluded that the extinction of organic radical intermediates results in inactivation of the enzyme by these coenzyme analogs. Upon suicide inactivation with a [15N2]imidazolyl analog, the octet signals due to Co(II) showed superhyperfine splitting into doublets, indicating axial coordination of 5, 6-dimethylben-zimidazole to the cobalamin bound to diol dehydratase. [ABSTRACT FROM AUTHOR]