Substrate specificity of coenzyme B12-dependent diol dehydrase: Glycerol as both a good substrate and a potent inactivator

Toraya, Tetsuo; Shirakashi, Tadahiro; Kosuga, Tsunemitsu; Fukui, Saburô

doi:10.1016/0006-291x(76)90546-5

Cited by 135 publications

(136 citation statements)

References 7 publications

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“…-dependent conversion of 1,2-propanediol, glycerol, and 1,2-ethanediol to the corresponding aldehydes (1,2). This enzyme is inducibly formed by some genera of Enterobacteriaceae, such as Klebsiella and Citrobacter, and other bacteria when they are grown anaerobically in medium containing 1,2-propanediol (3,4).…”

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confidence: 99%

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A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

Toraya

Mori

1999

Journal of Biological Chemistry

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Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing the ddrAB genes. They existed as a tight complex, i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with M r of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A 2 B 2 . The factor not only reactivated glycerol-inactivated and O 2 -inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg 2؉ . The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg 2؉, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzymebound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.Diol dehydratase (propane-1,2-diol hydro-lyase, EC 4.2.1.28) is an enzyme that catalyzes the adenosylcobalamin (AdoCbl)

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“…Klebsiella oxytoca ATCC 8724 is defective in glycerol dehydratase (10,11), but still capable of fermenting glycerol. This is because a low level of diol dehydratase induced by glycerol substitutes for isofunctional glycerol dehydratase (2,7,12,13).…”

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A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

Toraya

Mori

1999

Journal of Biological Chemistry

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“…Hence, it is likely that the rate of decomposition of acyl-Cbl is determined by the stability, rather than the abundance ratio, of its hydrated form. The assumption that the -CHO group in formyl-Cbl exists mainly in the hydrated form as in formaldehyde would be reasonable, because the electron-withdrawing effect or electronegativity of the Cbl moiety seems to be almost comparable to that of a hydrogen atom, as judged from similar chemical shifts of 19 F in CF3-Cbl and CF3H (17).…”

Section: Thermal Stability In Neutral Solution and In Acidic Solutionmentioning

confidence: 99%

“…We recently discussed about the mechanism of inactivation of AdoCbl-dependent diol dehydratase by glycerol (18,19) and the possible involvement of formyl-Cbl in the inactivation process (20). Theoretical calculations suggest that glycerol inactivation of this enzyme begins with an intramolecular hydrogen transfer from the glycerol 3-OH group to C1 of the substrate radical, forming an O3-centered radical that decomposes to formaldehyde and a glycol radical (20).…”

Section: Stability In Alkaline Solutionmentioning

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Synthesis and Properties of Formylcobalamin and Propionylcobalamin, Novel Acylcobalamins

Toraya

Ohya

2015

J Nutr Sci Vitaminol

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Summary Formylcobalamin (formyl-Cbl), a C1-unit carrying corrinoid, and propionylcobalamin (propionyl-Cbl) were synthesized for the fi rst time, and their properties were compared with those of acetylcobalamin (acetyl-Cbl). Formyl-Cbl, acetyl-Cbl, and propionyl-Cbl were decomposed by a NH2OH treatment, forming formo-, aceto-, and propionohydroxamic acids, respectively, which offers a proof for the presence of "activated" acyl groups and for their structures of Co-acyl-Cbls. These results, together with chromatographic, electrophoretic, and spectroscopic properties, indicate that the acyl-Cbls synthesized are actually formyl-Cbl, acetyl-Cbl, and propionyl-Cbl. Spectroscopic and electrophoretic properties were consistent with the -donor strength or trans-effect increasing in the order: formylϽacetylϽpropionyl. All acyl-Cbls underwent the Co-C bond cleavage upon photoirradiation with a tungsten light bulb as well as upon treatment with alkali, forming aquacobalamin (aqCbl) and hydroxocobalamin (OH-Cbl), respectively, under air. Formyl-Cbl was thermally unstable and decomposed to aqCbl, and the thermal stability in neutral solution was much lower than in diluted HCl. In contrast, acetyl-Cbl and propionyl-Cbl were heatstable under these conditions. Formyl-Cbl was essentially unsusceptible to unbufferized KCN solution, although acetyl-Cbl and propionyl-Cbl were rapidly converted to dicyanocobalamin under the same conditions. Such abnormal behaviors of formyl-Cbl suggest that it mainly exists in a hydrated form as formaldehyde.

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“…dependent conversion of 1,2-diols to the corresponding deoxy aldehydes (1)(2)(3). They consist of the three different subunits, ␣ ( Mr 60,000-61,000), ␤ ( Mr 21,000-24,000), and ␥ ( Mr 16,000-19,000), and dissociate into two dissimilar protein components in the absence of substrate ( 4,5 ).…”

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Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

Sakai

Yamasaki

Toyofuku

et al. 2007

J Nutr Sci Vitaminol

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Summary Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common ( ␣␤␥ ) 2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the ␣ subunit from glycerol dehydratase and the ␤ subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their ␣ subunits. Key Words coenzyme B 12 , adenosylcobalamin, diol dehydratase, glycerol dehydratase, hybrid enzymes Diol dehydratase ( DL -1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (EC 4.2.1.30) are isofunctional enzymes that catalyze the AdoCbl 1 -dependent conversion of 1,2-diols to the corresponding deoxy aldehydes ( 1-3 ). They consist of the three different subunits, ␣ ( Mr 60,000-61,000), ␤ ( Mr 21,000-24,000), and ␥ ( Mr 16,000-19,000), and dissociate into two dissimilar protein components in the absence of substrate ( 4, 5 ). Large and small components correspond to the ␣ 2 ␥ 2 complex and the ␤ subunit, respectively ( 6, 7 ). Their catalytic properties are similar, but they are different in the binding affinity for AdoCbl ( 8,9 ), substrate specificity ( 1-3, 10, 11 ), susceptibility to suicide inactivation by glycerol ( 2, 9, 10 ), monovalent cation specificity ( 1,11,12 ), and immunochemical reactivity toward anti-diol dehydratase antiserum ( 11 ) (for review, see Refs. 13-15 )). In order to reveal their similarities and differences on a molecular level, we cloned, sequenced, and expressed the diol dehydratase genes of Klebsiella oxytoca ( 16 ) and K lebsiella pneumoniae ( 17 ) and the glycerol dehydratase genes of K. pneumoniae ( 18 ). The glycerol dehydratase genes of Citrobacter freundii ( 19 ) and Clostridium pasteurianum ( 20 ) and the diol dehydratase genes of Salmonella typhimurium ( 21 ) and Lactobacillus collinoides ( 22 ) have also been cloned by other investigators. Comparison of the deduced amino acid sequences of the corresponding subunits between diol and glycerol dehydratases indicated that the ␣ subunit is most highly conserved among the subunits of the enzymes (identity, 71%), whereas the homologies of ␤ and ␥ subunits are lower (identities, 58% and 54%, respectively) ( Fig. 1) ( 18 ). The N-terminal 32 and 37 amino acid residues of the ␤ and ␥ subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase (16)(17)(18). These regions ...

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Substrate specificity of coenzyme B12-dependent diol dehydrase: Glycerol as both a good substrate and a potent inactivator

Cited by 135 publications

References 7 publications

A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

Synthesis and Properties of Formylcobalamin and Propionylcobalamin, Novel Acylcobalamins

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

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