Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism

Mancia, Filippo; Evans, Philip R.

doi:10.1016/s0969-2126(98)00073-2

Cited by 197 publications

(258 citation statements)

References 25 publications

(35 reference statements)

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“…We believe that the cleavage of AdoCbl is nonenzymatic and is due to photoreduction of the C-Co bond in the x-ray beam, a phenomenon reported for other AdoCbl enzymes (35). In the structure of 5,6-LAM, AdoH adopts a syn conformation about the glycosidic bond, in contrast to GM (36) and MCM (37), where the adenine ring of Ado is anti to the ribose ring. Here, AdoH is forced to adopt the syn conformation because of a steric clash between the adenine ring and Tyr-193␣ that would arise if AdoH were in the anti conformation.…”

Section: Resultsmentioning

confidence: 67%

“…There is precedent in the Cbl enzyme literature for a large-scale conformational change upon substrate binding (32,37,39). For methylcobalamin-dependent methionine synthase, the enzyme exists as an ensemble of conformational states that interconvert upon substrate or product binding (39).…”

Section: Discussionmentioning

confidence: 99%

“…X-ray analysis reveals that the two active sites that alternatively methylate and demethylate the Cbl cofactor are Ϸ50 Å apart (32), requiring large conformational rearrangements of the enzyme during each catalytic cycle. For AdoCbl-bound MCM, the presence of substrate has a dramatic effect on the structure of the TIM barrel (37). In the x-ray structure of the substratefree form of MCM, the TIM barrel is pried apart, leaving a large gap in the center of the barrel (37).…”

Section: Discussionmentioning

confidence: 99%

“…For AdoCbl-bound MCM, the presence of substrate has a dramatic effect on the structure of the TIM barrel (37). In the x-ray structure of the substratefree form of MCM, the TIM barrel is pried apart, leaving a large gap in the center of the barrel (37). Substrate binds in this gap, threading through the N terminus of the TIM barrel to the Cbl cofactor, which is located at the C terminus of the barrel (20).…”

Section: Discussionmentioning

confidence: 99%

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A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

109

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Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

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Section: Resultsmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Berkovitch

Behshad

Tang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

109

View full text Add to dashboard Cite

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“…Our own N-layered integrated molecular orbital and molecular mechanics (ONIOM) calculations using a recently developed optimization algorithm that allows the reaction coordinate to include atoms from both QM and molecular mechanics (MM) layers have permitted for the first time the calculation of the transition state (TS) for the enzyme-catalysed homolysis reaction (Kwiecien et al 2006). Three models, based on the crystal structures of the enzyme in the presence and absence of substrate (Mancia et al 1996(Mancia et al , 1999Mancia & Evans 1998), were employed in this Quantum catalysis in MCM R. Banerjee and others 1335 study. In the absence of substrate, the triosephosphate isomerase (TIM) barrel located above the corrin ring is flared open in a conformation that is referred to as the 'open' or resting state.…”

Section: Computational Analysis Of the Homolysis Reactionmentioning

confidence: 99%

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights

Banerjee

Dybała-Defratyka

Paneth

2006

Phil. Trans. R. Soc. B

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B 12 -dependent methylmalonyl-CoA mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt-carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 8C to 49.9 at 5 8C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt-carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt-carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.

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Glutamate Mutase

Kratky¹,

Gruber²

2004

Handbook of Metalloproteins

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The 5′‐adenosylcobalamin–dependent glutamate mutase catalyzes the reversible and stereospecific equilibration of ( S )‐glutamate with (2S,3S)‐3‐methylaspartate through a radical mechanism, whose first step consists of homolysis of the cofactor's cobalt–carbon bond. The enzyme occurs in clostridia, where it catalyzes the first step of the fermentation of glutamate. The protein exists as a heterotetramer of composition ɛ 2 σ 2 (B 12 ) 2 , with each of the two B 12 cofactor molecules located between an ɛ and a σ peptide. There are two substrate‐binding sites per heterotetramer at a distance of about 6 Å from the B 12 cofactor. The σ peptide folds as an α/β domain and is very similar to the B 12 ‐binding domains of other B 12 ‐dependent enzymes. The ɛ subunit consists of a (β/α) 8 ‐barrel (TIM‐barrel) domain, whose one end packs against the ‘upper’ face of the B 12 cofactor, thus forming the active site cavity.

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Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism

Cited by 197 publications

References 25 publications

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights

Glutamate Mutase

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Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism

Cited by 197 publications

References 25 publications

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Quantum catalysis in B 12 -dependent methylmalonyl-CoA mutase: experimental and computational insights

Glutamate Mutase

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Product

Resources

About

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights