Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase

Koichiro Kinoshita, Masahiro Kawata, Ken Ichi Ogura, Ai Yamasaki, Takeshi Watanabe, Noriaki Komoto, Naoki Hieda, Mamoru Yamanishi, Takamasa Tobimatsu, Tetsuo Toraya

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Abstract

Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisα143. Mutant enzymes in which Hisα143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Hα143Q mutant was 34% as active as the wild-type enzyme. Hα143A and Hα143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue α143 is important not only for catalysis but also for protecting radical intermediates. Hα143E and Hα143K that did not exist as (αβγ)2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Hα143Q and partially rate-determining for Hα143A. The preference for substrate enantiomers was reversed by the Hα143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Hα143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5′-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Hα143Q underwent irreversible inactivation by O2 in the absence of substrate at a much lower rate than the wild type.

Original languageEnglish
Pages (from-to)3162-3173
Number of pages12
JournalBiochemistry
Volume47
Issue number10
DOIs
Publication statusPublished - Mar 11 2008

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Propanediol Dehydratase
Holoenzymes
Catalysis
Histidine
Hydroxyl Radical
Hydrogen
Klebsiella oxytoca
Apoenzymes
Propylene Glycol
Deuterium
Coenzymes
Substrates
Enzymes
Hydrogen Bonding
Isotopes
X-Rays
Escherichia coli
Amino Acids
Mutation
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinoshita, K., Kawata, M., Ogura, K. I., Yamasaki, A., Watanabe, T., Komoto, N., ... Toraya, T. (2008). Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase. Biochemistry, 47(10), 3162-3173. https://doi.org/10.1021/bi7018095

Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase. / Kinoshita, Koichiro; Kawata, Masahiro; Ogura, Ken Ichi; Yamasaki, Ai; Watanabe, Takeshi; Komoto, Noriaki; Hieda, Naoki; Yamanishi, Mamoru; Tobimatsu, Takamasa; Toraya, Tetsuo.

In: Biochemistry, Vol. 47, No. 10, 11.03.2008, p. 3162-3173.

Research output: Contribution to journalArticle

Kinoshita, K, Kawata, M, Ogura, KI, Yamasaki, A, Watanabe, T, Komoto, N, Hieda, N, Yamanishi, M, Tobimatsu, T & Toraya, T 2008, 'Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase', Biochemistry, vol. 47, no. 10, pp. 3162-3173. https://doi.org/10.1021/bi7018095
Kinoshita, Koichiro ; Kawata, Masahiro ; Ogura, Ken Ichi ; Yamasaki, Ai ; Watanabe, Takeshi ; Komoto, Noriaki ; Hieda, Naoki ; Yamanishi, Mamoru ; Tobimatsu, Takamasa ; Toraya, Tetsuo. / Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase. In: Biochemistry. 2008 ; Vol. 47, No. 10. pp. 3162-3173.
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AU - Kinoshita, Koichiro

AU - Kawata, Masahiro

AU - Ogura, Ken Ichi

AU - Yamasaki, Ai

AU - Watanabe, Takeshi

AU - Komoto, Noriaki

AU - Hieda, Naoki

AU - Yamanishi, Mamoru

AU - Tobimatsu, Takamasa

AU - Toraya, Tetsuo

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N2 - Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisα143. Mutant enzymes in which Hisα143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Hα143Q mutant was 34% as active as the wild-type enzyme. Hα143A and Hα143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue α143 is important not only for catalysis but also for protecting radical intermediates. Hα143E and Hα143K that did not exist as (αβγ)2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Hα143Q and partially rate-determining for Hα143A. The preference for substrate enantiomers was reversed by the Hα143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Hα143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5′-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Hα143Q underwent irreversible inactivation by O2 in the absence of substrate at a much lower rate than the wild type.

AB - Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisα143. Mutant enzymes in which Hisα143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Hα143Q mutant was 34% as active as the wild-type enzyme. Hα143A and Hα143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue α143 is important not only for catalysis but also for protecting radical intermediates. Hα143E and Hα143K that did not exist as (αβγ)2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Hα143Q and partially rate-determining for Hα143A. The preference for substrate enantiomers was reversed by the Hα143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Hα143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5′-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Hα143Q underwent irreversible inactivation by O2 in the absence of substrate at a much lower rate than the wild type.

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