Abstract
Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+. Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg2+. Upon complex formation, one β subunit was released from the (αβ)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD β subunit and the reactivase β subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.
| Original language | English |
|---|---|
| Pages (from-to) | 4931-4943 |
| Number of pages | 13 |
| Journal | FEBS Journal |
| Volume | 277 |
| Issue number | 23 |
| DOIs | |
| Publication status | Published - Dec 2010 |
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Keywords
- Adenosylcobalamin
- Coenzyme B
- Diol dehydratase
- Diol dehydratase-reactivating factor
- Reactivase
ASJC Scopus subject areas
- Biochemistry
- Cell Biology
- Molecular Biology
Cite this
Diol dehydratase-reactivating factor is a reactivase - evidence for multiple turnovers and subunit swapping with diol dehydratase. / Mori, Koichi; Hosokawa, Yasuhiro; Yoshinaga, Toshiyuki; Toraya, Tetsuo.
In: FEBS Journal, Vol. 277, No. 23, 12.2010, p. 4931-4943.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Diol dehydratase-reactivating factor is a reactivase - evidence for multiple turnovers and subunit swapping with diol dehydratase
AU - Mori, Koichi
AU - Hosokawa, Yasuhiro
AU - Yoshinaga, Toshiyuki
AU - Toraya, Tetsuo
PY - 2010/12
Y1 - 2010/12
N2 - Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+. Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg2+. Upon complex formation, one β subunit was released from the (αβ)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD β subunit and the reactivase β subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.
AB - Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+. Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg2+. Upon complex formation, one β subunit was released from the (αβ)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD β subunit and the reactivase β subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.
KW - Adenosylcobalamin
KW - Coenzyme B
KW - Diol dehydratase
KW - Diol dehydratase-reactivating factor
KW - Reactivase
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UR - http://www.scopus.com/inward/citedby.url?scp=78449285232&partnerID=8YFLogxK
U2 - 10.1111/j.1742-4658.2010.07898.x
DO - 10.1111/j.1742-4658.2010.07898.x
M3 - Article
VL - 277
SP - 4931
EP - 4943
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 23
ER -