Roles of adenine anchoring and ion pairing at the coenzyme B 12-binding site in diol dehydratase catalysis

Ken Ichi Ogura, Shin Ichi Kunita, Koichi Mori, Takamasa Tobimatsu, Tetsuo Toraya

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sα224A and Sα224N mutants showed a k cat/kinact value that was less than 2% that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25-75%, except for the Kβ135E mutant (7%). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

Original languageEnglish
Pages (from-to)6204-6216
Number of pages13
JournalFEBS Journal
Volume275
Issue number24
DOIs
Publication statusPublished - Dec 2008

Fingerprint

Propanediol Dehydratase
Holoenzymes
Adenine
Vitamin B 12
Catalysis
Binding Sites
Ions
Enzymes
Apoenzymes
Enzyme Activation
Hydrogen Bonding
Cobalt
Amides
Cats
Carbon
Salts
Phosphates
X-Rays
Oxygen
Paramagnetic resonance

Keywords

  • Adenine anchoring
  • Adenosylcobalamin
  • Coenzyme B
  • Diol dehydratase
  • Ion pairing

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Roles of adenine anchoring and ion pairing at the coenzyme B 12-binding site in diol dehydratase catalysis. / Ogura, Ken Ichi; Kunita, Shin Ichi; Mori, Koichi; Tobimatsu, Takamasa; Toraya, Tetsuo.

In: FEBS Journal, Vol. 275, No. 24, 12.2008, p. 6204-6216.

Research output: Contribution to journalArticle

@article{f75f92f81b0f4ae499b6adebc46b00d4,
title = "Roles of adenine anchoring and ion pairing at the coenzyme B 12-binding site in diol dehydratase catalysis",
abstract = "The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19-2{\%} as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58-76{\%} of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sα224A and Sα224N mutants showed a k cat/kinact value that was less than 2{\%} that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25-75{\%}, except for the Kβ135E mutant (7{\%}). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.",
keywords = "Adenine anchoring, Adenosylcobalamin, Coenzyme B, Diol dehydratase, Ion pairing",
author = "Ogura, {Ken Ichi} and Kunita, {Shin Ichi} and Koichi Mori and Takamasa Tobimatsu and Tetsuo Toraya",
year = "2008",
month = "12",
doi = "10.1111/j.1742-4658.2008.06745.x",
language = "English",
volume = "275",
pages = "6204--6216",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "24",

}

TY - JOUR

T1 - Roles of adenine anchoring and ion pairing at the coenzyme B 12-binding site in diol dehydratase catalysis

AU - Ogura, Ken Ichi

AU - Kunita, Shin Ichi

AU - Mori, Koichi

AU - Tobimatsu, Takamasa

AU - Toraya, Tetsuo

PY - 2008/12

Y1 - 2008/12

N2 - The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sα224A and Sα224N mutants showed a k cat/kinact value that was less than 2% that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25-75%, except for the Kβ135E mutant (7%). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

AB - The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sα224A and Sα224N mutants showed a k cat/kinact value that was less than 2% that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25-75%, except for the Kβ135E mutant (7%). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

KW - Adenine anchoring

KW - Adenosylcobalamin

KW - Coenzyme B

KW - Diol dehydratase

KW - Ion pairing

UR - http://www.scopus.com/inward/record.url?scp=56849117882&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=56849117882&partnerID=8YFLogxK

U2 - 10.1111/j.1742-4658.2008.06745.x

DO - 10.1111/j.1742-4658.2008.06745.x

M3 - Article

C2 - 19016846

AN - SCOPUS:56849117882

VL - 275

SP - 6204

EP - 6216

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 24

ER -