Dynamic, Electrostatic Model for the Generation and Control of High‐Energy Radical Intermediates by a Coenzyme B<sub>12</sub>‐Dependent Enzyme

Chen, Huajun; Zietek, Monika; Russell, Henry J.; Tait, Shirley; Hay, Sam; Jones, Alex R.; Scrutton, Nigel S.

doi:10.1002/cbic.201300420

Cited by 14 publications

(11 citation statements)

References 37 publications

(34 reference statements)

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“…This decay involves a blue shift in the broad feature around 500–550 nm and a slight growth of the peak at ∼350 nm. Such relatively subtle spectral shifts are consistent with a change in the chromophore environment, with a blue shift suggesting it has become more hydrophobic (as observed for the chromophore in active-site variants of AdoCbl-dependent ethanolamine ammonia lyase) 29 . Consistent with the analysis of the ns–μs data is Co−C bond heterolysis to give five-coordinate cob(III)alamin and the 5′-deoxyadenosyl anion ( Fig.…”

Section: Resultssupporting

confidence: 69%

The photochemical mechanism of a B12-dependent photoreceptor protein

et al. 2015

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The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

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Section: Resultssupporting

confidence: 69%

The photochemical mechanism of a B12-dependent photoreceptor protein

et al. 2015

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“…In MCM and GM, a similar exchange of an active‐site glutamate residue was found to reduce catalytic turnover by 12‐ and 5000‐fold, respectively . Our findings are qualitatively similar to previous studies in OAM and other AdoCbl‐dependent enzymes in which electrostatic residues close to the cobalamin 2′ ribose hydroxyl were targeted . However, for reasons that are not clear, the extent of the loss in catalytic efficiency in the OAM variants reported here is less than that reported by Makins et al .…”

Section: Resultssupporting

confidence: 87%

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Menon

Fisher

et al. 2015

The FEBS Journal

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How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis.

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“…While the results of previous experimental mutagenesis studies [29][30][31] do not definitively prove that the QCE hypothesis is correct, they are certainly aligned with our findings. For example, it has been found for the GLM enzyme that replacing Glu330 with Asp or Gln results in decrease of tritium-exchange rate constants between Ado• and the substrate indicating larger hydrogen abstraction barrier heights for the mutants.…”

Section: Electronic Structures Of Glm and MCM Enzymessupporting

confidence: 77%

Radical Enzymes Control the Chemistry of Their Highly Reactive Intermediates Using the Quantum Coulombic Effect

Khalilian

DiLabio²

2020

Preprint

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Here, we report an exquisite strategy that the B12 enzymes exploit to manipulate the reactivity of their radical intermediate (Adenosyl radical). Based on the quantum-mechanic calculations, these enzymes utilize a little known long-ranged through space quantum Coulombic effect (QCE). The QCE causes the radical to acquire an electronic structure that contradicts the Aufbau Principle: The singly-occupied molecular orbital (SOMO) is no longer the highest-occupied molecular orbital (HOMO) and the radical is unable to react with neighbouring substrates. The dynamic nature of the enzyme and its structure is expected to be such that the reactivity of the radical is not restored until it is moved into close proximity of the target substrate. We found that the hydrogen bonding interaction between the nearby conserved glutamate residue and the ribose ring of Adenosyl radical plays a crucial role in manipulating the orbital ordering

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Dynamic, Electrostatic Model for the Generation and Control of High‐Energy Radical Intermediates by a Coenzyme B₁₂‐Dependent Enzyme

Cited by 14 publications

References 37 publications

The photochemical mechanism of a B12-dependent photoreceptor protein

The photochemical mechanism of a B12-dependent photoreceptor protein

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Radical Enzymes Control the Chemistry of Their Highly Reactive Intermediates Using the Quantum Coulombic Effect

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Dynamic, Electrostatic Model for the Generation and Control of High‐Energy Radical Intermediates by a Coenzyme B12‐Dependent Enzyme

Cited by 14 publications

References 37 publications

The photochemical mechanism of a B12-dependent photoreceptor protein

The photochemical mechanism of a B12-dependent photoreceptor protein

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Radical Enzymes Control the Chemistry of Their Highly Reactive Intermediates Using the Quantum Coulombic Effect

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Dynamic, Electrostatic Model for the Generation and Control of High‐Energy Radical Intermediates by a Coenzyme B₁₂‐Dependent Enzyme