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.
Highlights
Catalysis in ornithine 4,5-aminomutase (OAM) may involve large scale domain dynamics
Our data suggest that following ligand binding-induced cleavage of the Lys629-pyridoxal 5-phosphate (PLP) covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space
To reconcile the resting state structures obtained for OAM and related proteins with evidence for a short distance Co(II): substrate radical pair formed during catalysis, it was suggested these enzymes couple domain dynamics to radical formation [7, 11,12,13]
Summary
Catalysis in ornithine 4,5-aminomutase (OAM) may involve large scale domain dynamics. 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. Structural changes induced by ligand binding are modest in glutamate mutase/ethanolamine ammonia lyase, methylmalonyl-CoA mutase uses a large scale conformational change (closure of the TIM barrel) to trigger catalysis by coupling the local structural changes induced by the binding of a relatively large methylmalonyl-CoA substrate [16, 17] These changes are required to form a catalytically competent active site. Such a mechanism is clearly more simple to implement on a molecular level, and likely more robust, and is akin to the sampling of the flavin domain in electron-transferring flavoprotein complexes [21,22,23]
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