The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol

Mamoru Yamanishi, Michio Yunoki, Takamasa Tobimatsu, Hideaki Sato, Junko Matsui, Ayako Dokiya, Yasuhiro Iuchi, Kazunori Oe, Kyoko Suto, Naoki Shibata, Yukio Morimoto, Noritake Yasuoka, Tetsuo Toraya

Research output: Contribution to journalArticle

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Abstract

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2β2γ2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an iso-functional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

Original languageEnglish
Pages (from-to)4484-4494
Number of pages11
JournalEuropean Journal of Biochemistry
Volume269
Issue number18
DOIs
Publication statusPublished - 2002

Fingerprint

glycerol dehydratase
Propane
Propanediol Dehydratase
Vitamin B 12
Crystal structure
Isomers
Enzymes
Substrates
Catalytic Domain
Atoms
Enantiomers
Klebsiella pneumoniae
Crystallization
Cobalt
Hydroxyl Radical
Dimers
Glycerol
Suicide
Carrier concentration
cobamamide

Keywords

  • Adenosylcobalamin
  • Coenzyme B
  • Crystal structure
  • Glycerol dehydratase
  • Radical enzyme catalysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol. / Yamanishi, Mamoru; Yunoki, Michio; Tobimatsu, Takamasa; Sato, Hideaki; Matsui, Junko; Dokiya, Ayako; Iuchi, Yasuhiro; Oe, Kazunori; Suto, Kyoko; Shibata, Naoki; Morimoto, Yukio; Yasuoka, Noritake; Toraya, Tetsuo.

In: European Journal of Biochemistry, Vol. 269, No. 18, 2002, p. 4484-4494.

Research output: Contribution to journalArticle

Yamanishi, M, Yunoki, M, Tobimatsu, T, Sato, H, Matsui, J, Dokiya, A, Iuchi, Y, Oe, K, Suto, K, Shibata, N, Morimoto, Y, Yasuoka, N & Toraya, T 2002, 'The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol', European Journal of Biochemistry, vol. 269, no. 18, pp. 4484-4494. https://doi.org/10.1046/j.1432-1033.2002.03151.x
Yamanishi, Mamoru ; Yunoki, Michio ; Tobimatsu, Takamasa ; Sato, Hideaki ; Matsui, Junko ; Dokiya, Ayako ; Iuchi, Yasuhiro ; Oe, Kazunori ; Suto, Kyoko ; Shibata, Naoki ; Morimoto, Yukio ; Yasuoka, Noritake ; Toraya, Tetsuo. / The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol. In: European Journal of Biochemistry. 2002 ; Vol. 269, No. 18. pp. 4484-4494.
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AU - Tobimatsu, Takamasa

AU - Sato, Hideaki

AU - Matsui, Junko

AU - Dokiya, Ayako

AU - Iuchi, Yasuhiro

AU - Oe, Kazunori

AU - Suto, Kyoko

AU - Shibata, Naoki

AU - Morimoto, Yukio

AU - Yasuoka, Noritake

AU - Toraya, Tetsuo

PY - 2002

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N2 - Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2β2γ2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an iso-functional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

AB - Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2β2γ2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an iso-functional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

KW - Adenosylcobalamin

KW - Coenzyme B

KW - Crystal structure

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