A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes

Binuraj R. K. Menon, Karl Fisher, Stephen E. J. Rigby, Nigel S. Scrutton*, David Leys

*Corresponding author for this work

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Cobalamin-dependent enzymes enhance the rate of C–Co bond cleavage by up to ∼1012-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5′-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys629-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.
Original languageEnglish
Pages (from-to)34161-34174
Number of pages14
JournalJournal of Biological Chemistry
Issue number49
Early online date11 Sep 2014
Publication statusPublished - 5 Dec 2014


  • Adenosylcobalamin (AdoCbl)
  • Electron Paramagnetic Resonance (EPR)
  • protein dynamic
  • pyridoxal phosphate
  • radical
  • conformational sampling
  • domain dynamics
  • ornithine aminomutase


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