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

Mamoru Yamanishi, Koichiro Kinoshita, Masaki Fukuoka, Takuya Saito, Aya Tanokuchi, Yuuki Ikeda, Hirokazu Obayashi, Koichi Mori, Naoki Shibata, Takamasa Tobimatsu, Tetsuo Toraya

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 2OH 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. Structured digital abstract, and by () Coenzyme B 12-dependent diol dehydratase undergoes mechanism-based inactivation by glycerol. The X-ray structure indicated that glycerol is bound in the same conformation as that of (S)-1,2-propanediol. Certain mutants were substantially more resistant to the glycerol inactivation than the wild-type enzyme, with which the degree of preference for (S)-1,2-propanediol decreased. Substrate activities towards longer-chain 1,2-diols significantly increased with a double mutant.

Original languageEnglish
Pages (from-to)793-804
Number of pages12
JournalFEBS Journal
Volume279
Issue number5
DOIs
Publication statusPublished - Mar 2012

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Propanediol Dehydratase
Glycerol
Propylene Glycol
Conformations
Hydrogen
Enzymes
X-Rays
7-mercaptoheptanoylthreonine phosphate
Databases
X rays
Mutation
Coenzymes
Substrates
Amino Acid Substitution
Vitamin B 12

Keywords

  • adenosylcobalamin
  • coenzyme B
  • diol dehydratase
  • mechanism-based inactivation
  • redesign of enzyme
  • X-ray structure

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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. / Yamanishi, Mamoru; Kinoshita, Koichiro; Fukuoka, Masaki; Saito, Takuya; Tanokuchi, Aya; Ikeda, Yuuki; Obayashi, Hirokazu; Mori, Koichi; Shibata, Naoki; Tobimatsu, Takamasa; Toraya, Tetsuo.

In: FEBS Journal, Vol. 279, No. 5, 03.2012, p. 793-804.

Research output: Contribution to journalArticle

Yamanishi, Mamoru ; Kinoshita, Koichiro ; Fukuoka, Masaki ; Saito, Takuya ; Tanokuchi, Aya ; Ikeda, Yuuki ; Obayashi, Hirokazu ; Mori, Koichi ; Shibata, Naoki ; Tobimatsu, Takamasa ; Toraya, Tetsuo. / 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. In: FEBS Journal. 2012 ; Vol. 279, No. 5. pp. 793-804.
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abstract = "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 2OH 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. Structured digital abstract, and by () Coenzyme B 12-dependent diol dehydratase undergoes mechanism-based inactivation by glycerol. The X-ray structure indicated that glycerol is bound in the same conformation as that of (S)-1,2-propanediol. Certain mutants were substantially more resistant to the glycerol inactivation than the wild-type enzyme, with which the degree of preference for (S)-1,2-propanediol decreased. Substrate activities towards longer-chain 1,2-diols significantly increased with a double mutant.",
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AU - Yamanishi, Mamoru

AU - Kinoshita, Koichiro

AU - Fukuoka, Masaki

AU - Saito, Takuya

AU - Tanokuchi, Aya

AU - Ikeda, Yuuki

AU - Obayashi, Hirokazu

AU - Mori, Koichi

AU - Shibata, Naoki

AU - Tobimatsu, Takamasa

AU - Toraya, Tetsuo

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N2 - 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 2OH 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. Structured digital abstract, and by () Coenzyme B 12-dependent diol dehydratase undergoes mechanism-based inactivation by glycerol. The X-ray structure indicated that glycerol is bound in the same conformation as that of (S)-1,2-propanediol. Certain mutants were substantially more resistant to the glycerol inactivation than the wild-type enzyme, with which the degree of preference for (S)-1,2-propanediol decreased. Substrate activities towards longer-chain 1,2-diols significantly increased with a double mutant.

AB - 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 2OH 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. Structured digital abstract, and by () Coenzyme B 12-dependent diol dehydratase undergoes mechanism-based inactivation by glycerol. The X-ray structure indicated that glycerol is bound in the same conformation as that of (S)-1,2-propanediol. Certain mutants were substantially more resistant to the glycerol inactivation than the wild-type enzyme, with which the degree of preference for (S)-1,2-propanediol decreased. Substrate activities towards longer-chain 1,2-diols significantly increased with a double mutant.

KW - adenosylcobalamin

KW - coenzyme B

KW - diol dehydratase

KW - mechanism-based inactivation

KW - redesign of enzyme

KW - X-ray structure

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