Direct Determination of Product Radical Structure Reveals the Radical Rearrangement Pathway in a Coenzyme B<sub>12</sub>-Dependent Enzyme

Warncke, Kurt; Canfield, Jeffrey M.

doi:10.1021/ja031569d

Cited by 21 publications

(48 citation statements)

References 16 publications

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“…6. The first possible mechanism is similar to the working models of EAL 56,57 and diol dehydratase 10 catalysis and involves a radical-mediated 1,2-migration of the protonated amino group ( 28 → 29 ). The result would be a radical carbinolamine ( 29 ).…”

Section: Mechanism Of Deaminationmentioning

confidence: 84%

Mechanistic Enzymology of the Radical SAM Enzyme DesII

Ruszczycky

Liu

2015

Israel Journal of Chemistry

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DesII is a member of the radical SAM family of enzymes that catalyzes radical-mediated transformations of TDP-4-amino-4,6-didexoy-D-glucose as well as other sugar nucleotide diphosphates. Like nearly all radical SAM enzymes, the reactions begin with the reductive homolysis of SAM to produce a 5′-deoxyadenosyl radical which is followed by regiospecific hydrogen atom abstraction from the substrate. What happens next, however, depends on the nature of the substrate radical so produced. In the case of the biosynthetically relevant substrate, a radical-mediated deamination ensues; however, when this amino group is replaced with a hydroxyl, one instead observes dehydrogenation. The factors that govern the fate of the initially generated substrate radical as well as the mechanistic details underlying these transformations have been a key focus of research into the chemistry of DesII. This review will discuss recent discoveries pertaining to the enzymology of DesII, how it may relate to understanding other radical-mediated lyases and dehydrogenases and the working hypotheses currently being investigated regarding the mechanism of DesII catalysis.

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Section: Mechanism Of Deaminationmentioning

confidence: 84%

Mechanistic Enzymology of the Radical SAM Enzyme DesII

Ruszczycky

Liu

2015

Israel Journal of Chemistry

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“…Further, it is noted that the orientation of the C-N bond with respect to the radical center is preserved on going from reactant to product. 102 These results, however, do not distinguish between a stepwise (fragmentationrecombination) vs. concerted pathway for the migration.…”

Section: ð18þ ð19þmentioning

confidence: 88%

11 Reaction mechanisms : Part (i) Radical and radical ion reactions

Tanko

2005

Annu. Rep. Prog. Chem., Sect. B

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“…The reports (13, 20, 23) of a rearranged-substrate (carbinolamine) radical were surprising from the viewpoint of predicted stability of free radicals. In addition to the intrinsic lability of hemiaminals, the carbinolamine radical would have hyperconjugation to β substituents as its sole means of spin delocalization.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Substrate Radical Intermediate Derived from Ethanolamine during Catalysis by Ethanolamine Ammonia-Lyase

2008

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Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13 C]ethanolamine, [2-13 C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13 C hyperfine splitting in EPR spectra of samples prepared with [1-13 C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13 C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13 C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence for a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13 C hyperfine splitting tensor were analyzed through spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co 2+ to radical distance of 8.7Å was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13 C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of ∼98° with the unique axis of the d z 2 orbital of Co 2+ .Ethanolamine ammonia-lyase (EAL 1 , EC 4.3.1.7) is an AdoCbl dependent enzyme that catalyzes elimination of ammonia from the vicinal position of short chain amino-alcohols such as ethanolamine to give the corresponding oxo products. The functional protein is believed to be a hexamer of αβ-dimers ((αβ) 6 , α ∼50 kDa and β ∼31 kDa) (1,2). EAL is proposed to be an important enzyme in the metabolism of some bacterial species such as Salmonella enterica, which can use ethanolamine, derived from the breakdown of phospholipids, as their sole source † This research was supported by NIH Grant R56-GM35752. ‡ Present address: University of Michigan Medical School, 1150 W. Medical Center Dr., MSRB III, Ann Arbor, MI 48109-0606. * To whom correspondence should be addressed: University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Telephone: (608) 262-0509. Fax: (608) 265-2904. E-mail: ghreed@wisc.edu SUPPORTING INFORMATION AVAILABLE 13 C NMR spectra of ( 13 C)ethanolamines and 1 H NMR spectra of ( 13 C)glycine precursors. Stopped flow spectrophotometric assay for Co-carbon bond cleavage in the steady-state with ethanolamine. This material is available free of charge via the ...

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Direct Determination of Product Radical Structure Reveals the Radical Rearrangement Pathway in a Coenzyme B₁₂-Dependent Enzyme

Cited by 21 publications

References 16 publications

Mechanistic Enzymology of the Radical SAM Enzyme DesII

Mechanistic Enzymology of the Radical SAM Enzyme DesII

11 Reaction mechanisms : Part (i) Radical and radical ion reactions

Identification of the Substrate Radical Intermediate Derived from Ethanolamine during Catalysis by Ethanolamine Ammonia-Lyase

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Direct Determination of Product Radical Structure Reveals the Radical Rearrangement Pathway in a Coenzyme B12-Dependent Enzyme

Cited by 21 publications

References 16 publications

Mechanistic Enzymology of the Radical SAM Enzyme DesII

Mechanistic Enzymology of the Radical SAM Enzyme DesII

11 Reaction mechanisms : Part (i) Radical and radical ion reactions

Identification of the Substrate Radical Intermediate Derived from Ethanolamine during Catalysis by Ethanolamine Ammonia-Lyase

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Direct Determination of Product Radical Structure Reveals the Radical Rearrangement Pathway in a Coenzyme B₁₂-Dependent Enzyme