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 language | English |
|---|---|
| Pages (from-to) | 793-804 |
| Number of pages | 12 |
| Journal | FEBS Journal |
| Volume | 279 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Mar 2012 |
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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 journal › Article
}
TY - JOUR
T1 - 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
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
PY - 2012/3
Y1 - 2012/3
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
UR - http://www.scopus.com/inward/record.url?scp=84857655713&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84857655713&partnerID=8YFLogxK
U2 - 10.1111/j.1742-4658.2012.08470.x
DO - 10.1111/j.1742-4658.2012.08470.x
M3 - Article
C2 - 22221669
AN - SCOPUS:84857655713
VL - 279
SP - 793
EP - 804
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 5
ER -