Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases

Hideki Kajiura, Koichi Mori, Naoki Shibata, Tetsuo Toraya

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

Original languageEnglish
Pages (from-to)5556-5566
Number of pages11
JournalFEBS Journal
Volume274
Issue number21
DOIs
Publication statusPublished - Nov 2007

Fingerprint

glycerol dehydratase
Propanediol Dehydratase
Apoenzymes
Vitamin B 12
Enzymes
Adenosine Triphosphate
cobamamide
Holoenzymes

Keywords

  • Adenosylcobalamin
  • Coenzyme B
  • Diol dehydratase
  • Glycerol dehydratase
  • Reactivating factors

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases. / Kajiura, Hideki; Mori, Koichi; Shibata, Naoki; Toraya, Tetsuo.

In: FEBS Journal, Vol. 274, No. 21, 11.2007, p. 5556-5566.

Research output: Contribution to journalArticle

Kajiura, Hideki ; Mori, Koichi ; Shibata, Naoki ; Toraya, Tetsuo. / Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases. In: FEBS Journal. 2007 ; Vol. 274, No. 21. pp. 5556-5566.
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AB - Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

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