title: A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.
abstract: Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ∼10(12)-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 Lys(629)-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.
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Binuraj R K
authors.initials: BR
authors.lastname: Menon
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom nigel.scrutton@manchester.ac.uk.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom david.leys@manchester.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Leys
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D047991:Acetoanaerobium sticklandii / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000737:chemistry / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014805:Vitamin B 12 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D009952:Ornithine; D019751:Intramolecular Transferases; C403167:D-lysine 5,6-aminomutase; C021913:ornithine 5,4-aminomutase; D008239:Lysine; D014805:Vitamin B 12
keywords: Adenosylcobalamin (AdoCbl); Conformational Sampling; Domain Dynamics; Electron Paramagnetic Resonance (EPR); Ornithine Aminomutase; Protein Dynamic; Pyridoxal Phosphate; Radical