Identification of the 4-Glutamyl Radical as an Intermediate in the Carbon Skeleton Rearrangement Catalyzed by Coenzyme B12-Dependent Glutamate Mutase from Clostridium cochlearium

Bothe, Harald; Darley, Daniel J.; Albracht, S.P.J.; Gerfen, Gary J.; Golding, Bernard T.; Buckel, Wolfgañg

doi:10.1021/bi971393q

Cited by 98 publications

(128 citation statements)

References 33 publications

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“…This pseudorotation of the 5Ј-dAdo ribose group appears ideally suited to bridge the 5.5-6.5 Å distance between the substrates and the Cbl in IcmF. Other AdoCbl-dependent mutases similarly position their substrates at the same distance from the Cbl, as determined from crystal structures (24,29,33) and by electron paramagnetic resonance spectroscopic studies of glutamate mutase (62) and MCM (63) under catalytic conditions. Pseudorotation of the 5Ј-dAdo ribose has also been suggested from structural and biochemical studies of glutamate mutase (33,55) and from computational studies on MCM (64).…”

Section: Discussionmentioning

confidence: 88%

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

Jost

Born

Cracan

et al. 2015

Journal of Biological Chemistry

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Background: Acyl-CoA mutases catalyze radical-based carbon skeleton rearrangements. Results: Crystal structures of isobutyryl-CoA mutase in complex with four different substrates reveal active site architecture and determinants of substrate specificity. Conclusion: Identification of specificity-determining residues allows for prediction of new acyl-CoA mutase activities. Significance: Improved understanding of acyl-CoA mutase substrate specificity is critical for biotechnological and engineering applications.

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Section: Discussionmentioning

confidence: 88%

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

Jost

Born

Cracan

et al. 2015

Journal of Biological Chemistry

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“…The identity of the free radical partner to Cbl(II) formed in the reaction of 2-KG with glutamate mutase is of interest. Buckel and co-workers (21) have recently identified the C-4 radical of glutamate as the organic radical that accumulates on the enzyme during catalysis. Furthermore, we have demonstrated that, for both glutamate and methylaspartate, coenzyme homolysis and hydrogen abstraction are kinetically coupled (10).…”

Section: Discussionmentioning

confidence: 99%

The Reaction of the Substrate Analog 2-Ketoglutarate with Adenosylcobalamin-dependent Glutamate Mutase

Roymoulik

Chen

Marsh

1999

Journal of Biological Chemistry

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Glutamate mutase is one of several adenosylcobalamindependent enzymes that catalyze unusual rearrangements that proceed through a mechanism involving free radical intermediates. The enzyme exhibits remarkable specificity, and so far no molecules other than L-glutamate and L-threo-3-methylaspartate have been found to be substrates. Here we describe the reaction of glutamate mutase with the substrate analog, 2-ketoglutarate. Binding of 2-ketoglutarate (or its hydrate) to the holoenzyme elicits a change in the UV-visible spectrum consistent with the formation of cob(II)alamin on the enzyme. 2-ketoglutarate undergoes rapid exchange of tritium between the 5-position of the coenzyme and C-4 of 2-ketoglutarate, consistent with the formation of a 2-ketoglutaryl radical analogous to that formed with glutamate. Under aerobic conditions this leads to the slow inactivation of the enzyme, presumably through reaction of free radical species with oxygen. Despite the formation of a substrate-like radical, no rearrangement of 2-ketoglutarate to 3-methyloxalacetate could be detected. The results indicate that formation of the C-4 radical of 2-ketoglutarate is a facile process but that it does not undergo further reactions, suggesting that this may be a useful substrate analog with which to investigate the mechanism of coenzyme homolysis.Glutamate mutase catalyzes the reversible isomerization of L-glutamate and L-threo-3-methylaspartate (1-3). It is one of a group of enzymes that use adenosylcobalamin (AdoCbl), 1 a biologically active form of vitamin B 12 , to catalyze unusual 1,2-rearrangements in which an electron-withdrawing group is interchanged with a hydrogen atom on an adjacent carbon (4 -6) The migrating group may be -OH, -NH 2 , or, as in the case of glutamate mutase, a carbon-containing fragment so that a skeletal rearrangement is effected (Fig. 1). The role of AdoCbl as the source of carbon-based radicals, which are unmasked by homolysis of the coenzyme cobalt-carbon bond, is well established (7). Experiments with isotopically labeled substrates and coenzyme have demonstrated that for all the enzymes examined including glutamate mutase, 5Ј-deoxyadenosine acts as the intermediate carrier of the migrating hydrogen (8, 9).Recently, we have shown that in glutamate mutase homolysis of the cobalt-carbon bond and hydrogen abstraction from the substrate are kinetically coupled processes (10). Adenosyl radical can therefore only be formed as a transient high energy species, or may not be formed at all if coenzyme homolysis and hydrogen abstraction are truly concerted processes. Similar findings have been reported for the related AdoCbl-dependent enzyme, methylmalonyl-CoA mutase (11). These unexpected observations have focused our attention on the role of the substrate in initiating free radical formation. A further intriguing and poorly understood aspect of these reactions is how the enzyme controls the rearrangement of substrate-radical to product-radical.In principle, these steps might be probed with substrates that give rise ...

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“…Such intermediates have been detected by EPR spectroscopy of several AdoCbl-dependent isomerases trapped in steady-state catalytic turnover (5)(6)(7)(8). Effects of electron-electron spin coupling typically dominate the EPR spectra of these trapped intermediates (9).…”

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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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Identification of the 4-Glutamyl Radical as an Intermediate in the Carbon Skeleton Rearrangement Catalyzed by Coenzyme B₁₂-Dependent Glutamate Mutase from Clostridium cochlearium

Cited by 98 publications

References 33 publications

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

The Reaction of the Substrate Analog 2-Ketoglutarate with Adenosylcobalamin-dependent Glutamate Mutase

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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