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A reactivating factor for coenzyme B12-dependent diol dehydratase

T Toraya1, K Mori

  • 1Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan. toraya@biotech.okayama-u.ac.jp

The Journal of Biological Chemistry
|January 28, 1999
PubMed
Summary

Diol dehydratase inactivation by glycerol is reversed by a DdrAB protein complex. This complex exchanges bound cobalamin, reactivating the enzyme for crucial metabolic functions.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Adenosylcobalamin-dependent diol dehydratase in Klebsiella oxytoca is inactivated by its substrate, glycerol.
  • Inactivation involves irreversible cleavage of the coenzyme's cobalt-carbon bond, leading to tight binding within the enzyme's active site.

Purpose of the Study:

  • To investigate the mechanism of diol dehydratase inactivation and reactivation.
  • To characterize the putative reactivating factor (DdrA and DdrB proteins) from Klebsiella oxytoca.

Main Methods:

  • Co-purification of recombinant DdrA and DdrB proteins from Escherichia coli overexpressing ddrAB genes.
  • Determination of the molecular weight and subunit structure of the DdrAB complex.
  • Assays to test the reactivation of inactivated holoenzymes and activation of enzyme-cyanocobalamin complexes.

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Main Results:

  • A tight complex of DdrA and DdrB (putative reactivating factor) was purified, with an apparent molecular weight of 150,000 and an A2B2 subunit structure.
  • The DdrAB factor reactivated glycerol-inactivated and O2-inactivated holoenzymes.
  • The factor also activated an enzyme-cyanocobalamin complex in the presence of adenosylcobalamin, ATP, and Mg2+.
  • ATP-dependent exchange of enzyme-bound cyanocobalamin for adenosylcobalamin was mediated by the DdrAB factor.

Conclusions:

  • The DdrAB complex acts as a reactivating factor for inactivated diol dehydratase.
  • Reactivation occurs through the exchange of enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.
  • This mechanism highlights the dynamic nature of cobalamin binding in enzyme reactivation.