The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

Reed, George H.; Mansoorabadi, Steven O.

doi:10.1016/j.sbi.2003.10.011

Cited by 20 publications

(20 citation statements)

References 28 publications

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“…The positions of radical intermediates in the active sites were reviewed by Reed and Mansoorabadi [82]. The first direct proof of radical mechanism in these enzymes was obtained in the reaction of the substrate homologue 2,4-DAB with 4,5-OAM only five years back [70].…”

Section: Mechanistic Studiesmentioning

confidence: 99%

Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

Maity

Chen

2014

IJMS

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Lysine 5,6-aminomutase (5,6-LAM) and ornithine 4,5-aminomutase (4,5-OAM) are two of the rare enzymes that use assistance of two vitamins as cofactors. These enzymes employ radical generating capability of coenzyme B12 (5′-deoxyadenosylcobalamin, dAdoCbl) and ability of pyridoxal-5′-phosphate (PLP, vitamin B6) to stabilize high-energy intermediates for performing challenging 1,2-amino rearrangements between adjacent carbons. A large-scale domain movement is required for interconversion between the catalytically inactive open form and the catalytically active closed form. In spite of all the similarities, these enzymes differ in substrate specificities. 4,5-OAM is highly specific for d-ornithine as a substrate while 5,6-LAM can accept d-lysine and l-β-lysine. This review focuses on recent computational, spectroscopic and structural studies of these enzymes and their implications on the related enzymes. Additionally, we also discuss the potential biosynthetic application of 5,6-LAM.

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Section: Mechanistic Studiesmentioning

confidence: 99%

Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

Maity

Chen

2014

IJMS

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“…the low spin Co 2ϩ and the substrate/product intermediate) formed during turnover of AdoCbl-dependent enzymes is a function of their separation and relative orientation (10). Analysis of the EPR signatures from Class I and II isomerases reveals that the former have strong exchange-coupled spin systems, indicating an interspin distance of Ͻ6 Å (22,35,36), whereas the latter exhibit a weakly coupled spin system, reflecting a larger distance (11-13 Å) between the paramagnetic centers (30,37).…”

Section: Cob(ii)alamin Is Formed Only Transiently Upon Reactionmentioning

confidence: 99%

“…In the presence of substrate, cob(II)alamin accumulates at a steady-state concentration, which can be observed by EPR and UV-visible spectroscopy. Class I and Class II isomerases have been extensively studied, and the cob(II)alamin spectroscopic signature has been valuable in studies of mechanism (6,8,10). Lysine 5,6-aminomutase (5,6-LAM) is the only Class III enzyme for which detailed studies of mechanism are reported.…”

mentioning

confidence: 99%

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Wolthers

Rigby

Scrutton

2008

Journal of Biological Chemistry

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We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin ( ; coenzyme B 12 ) serves as a radical repository for a group of enzymes that catalyze unusual isomerizations, whereby a hydrogen atom (H) is interchanged with an electron-withdrawing group (X) on a neighboring carbon atom (Scheme 1) (1-4). To date, 11 AdoCbl-dependent isomerases have been identified and grouped into three classes: (i) Class I (mutases) that catalyze carbon skeletal rearrangements; (ii) Class II (eliminases) that catalyze, with one exception, the migration and elimination of a heteroatom; and (iii) Class III (aminomutases) that catalyze intramolecular 1,2-amino shifts. Turnover for all AdoCbl-dependent isomerases begins with substrate-induced homolysis of the AdoCbl Co-C bond and formation of two paramagnetic centers: the 5Ј-deoxyadenosyl radical (Ado ⅐ ) and cob(II)alamin. The highly reactive Ado ⅐ species propagates radical formation by abstracting a hydrogen atom from the substrate (or an amino acid side chain in the case of ribonucleotide reductase) (5), generating deoxyadenosine and a substrate radical. The latter carbon-centered radical isomerizes to a product radical intermediate, which then reabstracts a hydrogen atom to form Ado ⅐ . Geminate recombination between Ado ⅐ and cob(II)alamin regenerates AdoCbl and primes the enzyme for another catalytic cycle.Studies on AdoCbl-dependent isomerases have shown that the first step in the catalytic cycle (homolytic rupture of the Co-C bond) is coupled kinetically to hydrogen abstraction by Ado ⅐ (6 -9). Thus, the highly reactive Ado ⅐ species is short lived due to rapid neutralization by hydrogen abstraction, and this species has yet to be observed. The second paramagnetic center, cob(II)alamin, "lingers" in the active site, until product is formed, after which it recombines with Ado ⅐ to form the resting enzyme. In the presence of substrate, cob(II)alamin accumulates at a steady-state concentration, which can be observed by EPR and UV-visible spectroscopy. Class I and Class II isomerases have been extensively studied, and the cob(II)alamin spectroscopic signature has been valuable in studies of mechanism (6,8,10). Lysine 5,6-aminomutase (5,6-LAM) is the only Class III enzyme for which detailed studies of mechanism are reported. Cob(II)alamin does not accumulate in steady-state turnover as the enzyme undergoes rapid "suicide inactivation" involving removal of an electron from cob(II)alamin by a substrate and/or product radical intermediate (11). Irreversible formation of cob(III)alamin results, and cob(II)alamin is unable to recombine with Ado ⅐ to reform AdoCbl in the resting form of the enzyme.

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“…In particular, the apparent g values in the spectrum and the magnitude of the 59 Co hyperfine splitting are similar to those found for other cob(II)alamin-organic radical triplets in AdoCbl-dependent enzymes (9). The appearance of the EPR spectra in these hybrid triplet systems is dependent on the separation between Co 2+ and the organic radical and on the position of the organic radical in the g-axis system of Co 2+ (9,25,26). The radical rearrangement that is outlined in Figure 1 includes the primary alkyl radicals, the 5′-deoxyadenosyl radical and the methylmalonyl-CoA radical, and a secondary alkyl radical, the succinyl-CoA radical.…”

Section: Analysis Of the Epr Patternmentioning

confidence: 99%

Characterization of a Succinyl-CoA Radical−Cob(II)alamin Spin Triplet Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase

et al. 2005

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The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co 2+ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zerofield splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co 2+ . Labeling of the substrate with 13 C and 2 H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2′-13 C]methylmalonyl-CoA and [2-13 C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon α to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme. In all AdoCbl-dependent isomerases, the cobalamin cofactor functions as a radical initiator, and the ensuing isomerization reactions proceed through a series of radical intermediates (Figure 1). The first and common step, among all of the enzymes, is homolysis of the cobalt-carbon σ-bond of the cofactor to generate a pair of radicals, cob(II)alamin [which

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The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

Cited by 20 publications

References 28 publications

Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

Large-Scale Domain Motions and Pyridoxal-5'-Phosphate Assisted Radical Catalysis in Coenzyme B12-Dependent Aminomutases

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Characterization of a Succinyl-CoA Radical−Cob(II)alamin Spin Triplet Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase

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