Identification of a paramagnetic species as an early intermediate in the coenzyme B<sub>12</sub>‐dependent glutamate mutase reaction A cob(II)amide?

Leutbecher, Ulrich; Albracht, S.P.J.

doi:10.1016/0014-5793(92)80754-5

Cited by 38 publications

(31 citation statements)

References 12 publications

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“…In the presence of 2-methyleneglutarate or its competitive inhibitor itaconate, an additional EPR signal at g = 2.1 was detected in 2-methyleneglutarate mutase (Michel et al, 1992;Zelder and Buckel, 1993). Similar signals were also observed with the related adenosylcobalamin dependent enzymes glutamate mutase from Clostridium cochlearium (Leutbecher et al, 1992) and methylmalonyl-CoA mutase from Propionibacterium freudenreichii (Zhao et al, 1992). Especially the EPR spectra observed with glutamate mutase were interpreted as a co-ordination of the substrate with the cobalt during the rearrangement.…”

Section: Fig 1 Reactions Catalyzed By 2-methyleneglutarate Mutase Amentioning

confidence: 54%

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Beatrix

Zelder

Linder

et al. 1994

European Journal of Biochemistry

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The coenzyme B,, (adenosy1cobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri.(a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region.(b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/ PAGE and Westem-blot analysis with rabbit antiserum directed against the native mutase.(c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B,2. Experiments are presented which suggest that the expression product is an apoenzyme.

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Section: Fig 1 Reactions Catalyzed By 2-methyleneglutarate Mutase Amentioning

confidence: 54%

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Beatrix

Zelder

Linder

et al. 1994

European Journal of Biochemistry

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“…Free cob(I1)alamin is easily oxidized to aquocobalamin whereas enzyme-bound cob(II)almin is often oxygen stable. This was shown for the inactive glutamate mutase (Leutbecher et al, 1992b) and for 2-methyleneglutarate mutase (Zelder and Buckel, 1993). In the latter enzyme cob(1I)-alamin also represents an inactive enzyme-coenzyme complex.…”

Section: Inactivation Of Glutamate Mutase Under the Formation Of Cob(mentioning

confidence: 99%

“…Incubation of components E and S without coenzyme did not result in assembly of the subunits. Purification of glutamate mutase from C. cochlearium revealed pure and colourless components E and S (Leutbecher et al, 1992b) as well as an additional glutamate mutase fraction that consisted of both subunits, aquocobalamin and cob-(1I)alamin but did not show any enzyme activity. It was analysed as described above and it was also shown to be a heterotetramer containing lmol Co/mol e2o2.…”

Section: Coenzyme B Binding Studiesmentioning

confidence: 99%

“…The signal was tentatively identified as a novel Co(I1) species. The formation of the signal required less than 15 ms and its intensity did not change within a further 15 min (Leutbecher et al, 1992b). Now we have obtained EPR spectra of much better resolution clearly showing the involvement of Co(I1) (gx,, = 2.1, g, = 1.985, A, = 50 mT) (Fig.…”

Section: The Substrate-induced Epr Signal Probably Represents a Steadmentioning

confidence: 99%

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Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Zelder¹,

Beatrix²,

Leutbecher³

1994

European Journal of Biochemistry

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The glutamate mutase dependent on adenosylcobalamin (coenzyme B,J catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer E~ ( E , 53.5 kDa) and S, a monomer (a, 14.8 ma). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins.Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme BIZ. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B,, resulted in the formation of a fully active glutamate mutase heterotetramer (~~a~) containing one molecule of coenzyme BIZ.EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B,, and (S)-glutamate. The EPR signals (gx, = 2.1, g, = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(I1)alamin (gx,, = 2.25). The spin concentration was 34-50% of the concentration of the enzyme ( E~C T J coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)- [2,3,3,4,]glutaate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7 %. These data suggest an involvement of the Co(I1)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the perdeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species. It is discussed that the paramagnetic species represents a radical pair composed of Co(I1) and an organic radical.Overnight incubation of glutamate mutase and coenzyme with substrate or one of the competitive inhibitors resulted in an inactive enzyme and cob(II)alamin, which was identified by EPR spectroscopy (gX,, = 2.25). This cob(I1)alamin appeared to be very similar to a cob(I1)amide present in inactive glutamate mutase preparations...

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“…[8,13] The localisation of the carbon-centred radicals in the substrate/product has been experimentally determined for ethanolamine ammonia-lyase, [14] glutamate mutase, [15] and 2-methyleneglutarate mutase (A. J. Pierik, unpublished results) by EPR spectroscopy of the enzyme in the presence of isotopically-labelled substrates. These studies and the rapid-freeze experiments performed by Babior et al [14] and Leutbecher et al [16] exclude substantial concentrations of amino acid-centred and deoxyadenosyl radical species as intermediates. Attempts to detect the 5'-deoxyadenosyl radical have involved its stabilisation by the introduction of a double bond into the ribose ring.…”

Section: Introductionmentioning

confidence: 86%

Synthesis of Enantiomerically‐Pure [¹³C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions

Weigl¹,

Heimberger²,

Pierik³

et al. 2003

Chemistry A European J

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We describe a novel enantioselective synthesis of aristeromycin, the carbocyclic analogue of adenosine. The seven-step synthesis is also suitable for the preparation of specifically-labelled [6'-(13)C]aristeromycin. Both the unlabelled and (13)C-labelled product was coupled to vitamin B(12) to form aristeromycylcobalamin. This carbocyclic analogue of coenzyme B(12) was examined for its coenzymic activity with several adenosylcobalamin-dependent enzymes. For glyceroldehydratase and dioldehydratase, the reaction rate (k(cat)) was 38 and 44 % of that measured with adenosylcobalamin as coenzyme. In contrast, aristeromycylcobalamin showed no detectable activity with methylmalonyl-CoA mutase and ethanolamine ammonia-lyase. Instead, it was a weak inhibitor of the former and a strong inhibitor of the latter enzyme. The slower turnover rate with glyceroldehydratase raised the hope of detecting the 6'-deoxyaristeromycyl radical intermediate. Comparison of the EPR spectra of the intermediates in the glyceroldehydratase reaction, which used adenosyl- and aristeromycylcobalamines, respectively, as coenzyme, revealed a significant shift and this suggests a different geometric position of these cofactors at the binding site during the cleavage of the carbon-cobalt bond. However, we found no evidence for the existence of a 6'-deoxyaristeromycyl radical during the reaction with [6'-(13)C]aristeromycylcobalamin. We conclude that the lifetime of this radical is still too short to be observed.

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Identification of a paramagnetic species as an early intermediate in the coenzyme B₁₂‐dependent glutamate mutase reaction A cob(II)amide?

Cited by 38 publications

References 12 publications

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Synthesis of Enantiomerically‐Pure [¹³C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions

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Identification of a paramagnetic species as an early intermediate in the coenzyme B12‐dependent glutamate mutase reaction A cob(II)amide?

Cited by 38 publications

References 12 publications

Cloning, sequencing and expression of the gene encoding the coenzyme B12‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Cloning, sequencing and expression of the gene encoding the coenzyme B12‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Characterization of the Coenzyme‐B12–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Synthesis of Enantiomerically‐Pure [13C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions

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Identification of a paramagnetic species as an early intermediate in the coenzyme B₁₂‐dependent glutamate mutase reaction A cob(II)amide?

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Cloning, sequencing and expression of the gene encoding the coenzyme B₁₂‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Synthesis of Enantiomerically‐Pure [¹³C]Aristeromycylcobalamin and Its Reactivity in Dioldehydratase, Glyceroldehydratase, Ethanolamine Ammonia‐Lyase and Methylmalonyl‐CoA Mutase Reactions