Survey of catalytic residues and essential roles of glutamate-α170 and aspartate-α335 in coenzyme b12-dependent diol dehydratase

Masahiro Kawata, Koichiro Kinoshita, Sumihisa Takahashi, Ken Ichi Ogura, Noriaki Komoto, Mamoru Yamanishi, Takamasa Tobimatsu, Tetsuo Toraya

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)


The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K+ was mutated to Ala or another amino acid residue. The Eα170A and Dα335A mutants were totally inactive, and the Hα143A mutant showed only a trace of activity, indicating that Glu-α170, Asp-α335, and His-α143 are catalytic residues. The Qα141A, Qα296A, and Sα362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-α296 is important for substrate binding and Gln-α296 and Gln-α141 for preventing the enzyme from mechanism-based inactivation. The Eα221A, Eα170H, and Dα335A did not form the (αβγ)2 complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-α170 and Asp-α335 mutants, Eα170D and Eα170Q were 2.2 ± 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dα335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue α170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-α170 and Asp-α335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-α170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.

Original languageEnglish
Pages (from-to)18327-18334
Number of pages8
JournalJournal of Biological Chemistry
Issue number27
Publication statusPublished - Jul 7 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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