Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase

Tetsuo Toraya, Aya Tanokuchi, Ai Yamasaki, Takehiro Nakamura, Kenichi Ogura, Takamasa Tobimatsu

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg2+ (or Mn2+) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg2+, diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

Original languageEnglish
Pages (from-to)69-78
Number of pages10
JournalBiochemistry
Volume55
Issue number1
DOIs
Publication statusPublished - Jan 19 2016

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Propanediol Dehydratase
Recycling
Coenzymes
Holoenzymes
NAD
glycerol dehydratase
Adenosine Triphosphate
Klebsiella oxytoca
Hydro-Lyases
Reducing Agents
Vitamin B 12
Prosthetics
cobamamide
Complex Mixtures
Glycerol
Bacteria
Oxidoreductases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase. / Toraya, Tetsuo; Tanokuchi, Aya; Yamasaki, Ai; Nakamura, Takehiro; Ogura, Kenichi; Tobimatsu, Takamasa.

In: Biochemistry, Vol. 55, No. 1, 19.01.2016, p. 69-78.

Research output: Contribution to journalArticle

Toraya, Tetsuo ; Tanokuchi, Aya ; Yamasaki, Ai ; Nakamura, Takehiro ; Ogura, Kenichi ; Tobimatsu, Takamasa. / Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase. In: Biochemistry. 2016 ; Vol. 55, No. 1. pp. 69-78.
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