A Protein Factor Is Essential for in Situ Reactivation of Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase

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Abstract

The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. β,γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3′-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

Original languageEnglish
Pages (from-to)1729-1733
Number of pages5
JournalBioscience, Biotechnology and Biochemistry
Volume61
Issue number10
Publication statusPublished - Oct 1997

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Propanediol Dehydratase
Adenosinetriphosphate
Glycerol
coenzymes
glycerol
Coenzymes
Adenosine Triphosphate
Proteins
Holoenzymes
plasmids
proteins
Escherichia coli
Klebsiella oxytoca
Plasmids
Genes
ribose
3' Flanking Region
adenine
catalytic activity
Ribose

Keywords

  • Adenosylcobalamin
  • Diol dehydratase
  • Reactivating factor
  • Suicide inactivation
  • Vitamin B

ASJC Scopus subject areas

  • Food Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Chemistry (miscellaneous)
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

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abstract = "The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. β,γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3′-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.",
keywords = "Adenosylcobalamin, Diol dehydratase, Reactivating factor, Suicide inactivation, Vitamin B",
author = "Koichi Mori and Takamasa Tobimatsu and Tetsuo Toraya",
year = "1997",
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language = "English",
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T1 - A Protein Factor Is Essential for in Situ Reactivation of Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase

AU - Mori, Koichi

AU - Tobimatsu, Takamasa

AU - Toraya, Tetsuo

PY - 1997/10

Y1 - 1997/10

N2 - The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. β,γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3′-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

AB - The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. β,γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3′-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

KW - Adenosylcobalamin

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KW - Reactivating factor

KW - Suicide inactivation

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