Publications

• Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase.

  Brooks A.J., Fox C.C., Marsh E.N., Vlasie M., Banerjee R., Brunold T.C.

  Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an ... >> More

  Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an adenosyl radical and Co2+ Cbl. In this work, we employed a combination of spectroscopic and computational tools to evaluate possible mechanisms by which the Co-C bond is activated for homolysis. Minimal perturbations to the electronic absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even in the presence of substrate (or a substrate analogue), indicating that destabilization of the Co3+ Cbl "ground state" is an unlikely mechanism for Co-C bond activation. In contrast, striking alterations are observed in the spectroscopic data of the post-homolysis product Co2+ Cbl when bound to glutamate mutase in the presence of substrate (or a substrate analogue) as compared to unbound Co2+ Cbl. These enzymatic perturbations appear to most strongly affect the metal-to-ligand charge-transfer transitions of Co2+ Cbl, suggesting that the cofactor/active-site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals. Remarkable similarities between the results obtained in this study and those reported previously for the related Cbl-dependent isomerase methylmalonyl-CoA mutase indicate that a common mechanism by which the cofactor's Co-C bond is activated for homolytic cleavage may be operative for all base-off/His-on Cbl-dependent isomerases. << Less

  Biochemistry 44:15167-15181(2005) [PubMed] [EuropePMC]

• Radical shuttling in a protein: ribose pseudorotation controls alkyl-radical transfer in the coenzyme B(12) dependent enzyme glutamate mutase.

  Gruber K., Reitzer R., Kratky C.

  Angew. Chem. Int. Ed. Engl. 40:3377-3380(2001) [PubMed] [EuropePMC]

• Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli.

  Zelder O., Beatrix B., Leutbecher U., Buckel W.

  The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) 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 ... >> More

  The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) 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 epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). 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 B12. 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 B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 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(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) 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)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-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 predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS) << Less

  Eur. J. Biochem. 226:577-585(1994) [PubMed] [EuropePMC]

• Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme.

  Holloway D.E., Marsh E.N.

  The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli. This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS. MutE binds MutS ... >> More

  The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli. This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS. MutE binds MutS cooperatively, with a Hill coefficient of 1.3. The recombinant enzyme has an unchanged Km for L-glutamate, but a much higher specific activity than those previously reported for preparations from clostridia. The apparent Km for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 microM with equimolar concentrations of MutS and MutE and 5.8 microM with a 5-fold molar excess of MutS over MutE present in the assay. The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel filtration. In the presence of equimolar amounts of MutE and MutS, the apparent Kd was 5.4 microM, but this decreased to 1.8 microM when MutS was present at a 5-fold molar excess. No binding of adenosylcobalamin to MutE was observed in the absence of MutS. This suggests that the (minimal) function for MutS, whose role in the reaction has been unclear until now, is to form part of the adenosylcobalamin-binding site. It seems likely that MutS is representative of a cobalamin-binding domain conserved across several cobalamin-dependent enzymes. << Less

  J. Biol. Chem. 269:20425-20430(1994) [PubMed] [EuropePMC]

• Glutamate mutase from Clostridium cochlearium. Purification, cobamide content and stereospecific inhibitors.

  Leutbecher U., Boecher R., Linder D., Buckel W.

  Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50- ... >> More

  Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260-3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-[alpha-(aden-9-yl)]-cob(II)amide. A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy. A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis. Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM). (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained. << Less

  Eur. J. Biochem. 205:759-765(1992) [PubMed] [EuropePMC]

• Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights.

  Reitzer R., Gruber K., Jogl G., Wagner U.G., Bothe H., Buckel W., Kratky C.

  <h4>Background</h4>Glutamate mutase (Glm) equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. Catalysis proceeds with the homolytic cleavage of the organometallic bond of the cofactor to yield a 5'-desoxyadenosyl radical. This radical then abstracts a hydrogen atom from the protein-bound su ... >> More

  <h4>Background</h4>Glutamate mutase (Glm) equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. Catalysis proceeds with the homolytic cleavage of the organometallic bond of the cofactor to yield a 5'-desoxyadenosyl radical. This radical then abstracts a hydrogen atom from the protein-bound substrate to initiate the rearrangement reaction. Glm from Clostridium cochlearium is a heterotetrameric molecule consisting of two sigma and two epsilon polypeptide chains.<h4>Results</h4>We have determined the crystal structures of inactive recombinant Glm reconstituted with either cyanocobalamin or methylcobalamin. The molecule shows close similarity to the structure of methylmalonyl CoA mutase (MCM), despite poor sequence similarity between its catalytic epsilon subunit and the corresponding TIM-barrel domain of MCM. Each of the two independent B12 cofactor molecules is associated with a substrate-binding site, which was found to be occupied by a (2S,3S)-tartrate ion. A 1:1 mixture of cofactors with cobalt in oxidation states II and III was observed in both crystal structures of inactive Glm.<h4>Conclusions</h4>The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction. << Less

  Structure 7:891-902(1999) [PubMed] [EuropePMC]

• Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism.

  Marsh E.N., Drennan C.L.

  Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structure ... >> More

  Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structures of several of these enzymes have been solved, revealing two modes of coenzyme binding and highlighting the role of the protein in controlling the rearrangement of reactive substrate radical intermediates. Complementary data from kinetic, spectroscopic and theoretical studies have produced insights into the mechanism by which substrate radicals are generated at the active site, and the pathways by which they rearrange. << Less

  Curr Opin Chem Biol 5:499-505(2001) [PubMed] [EuropePMC]

• Interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate: a distinctive B(12)-dependent carbon-skeleton rearrangement.

  Wetmore S.D., Smith D.M., Golding B.T., Radom L.

  The interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate catalyzed by B(12)-dependent glutamate mutase is discussed using results from high-level ab initio molecular orbital calculations. Evidence is presented regarding the possible role of coenzyme-B(12) in substrate activation and prod ... >> More

  The interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate catalyzed by B(12)-dependent glutamate mutase is discussed using results from high-level ab initio molecular orbital calculations. Evidence is presented regarding the possible role of coenzyme-B(12) in substrate activation and product formation via radical generation. Calculated electron paramagnetic resonance parameters support experimental evidence for the involvement of substrate-derived radicals and will hopefully aid the future detection of other important radical intermediates. The height of the rearrangement barrier for a fragmentation-recombination pathway, calculated with a model that includes neutral amino and carboxylic acid substituents in the migrating glycyl group, supports recent experimental evidence for the interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate through such a pathway. Our calculations suggest that the enzyme may facilitate the rearrangement of (S)-glutamate through (partial) proton-transfer processes that control the protonation state of substituents in the migrating group. << Less

  J Am Chem Soc 123:7963-7972(2001) [PubMed] [EuropePMC]

• The role of the active site glutamate in the rearrangement of glutamate to 3-methylaspartate catalyzed by adenosylcobalamin-dependent glutamate mutase.

  Madhavapeddi P., Marsh E.N.

  <h4>Background</h4>Adenosylcobalamin (coenzyme B(12))-dependent enzymes catalyze a variety of chemically difficult reactions that proceed through the generation of free radical intermediates. A long-standing question is how proteins stabilize what are normally regarded as highly reactive organic r ... >> More

  <h4>Background</h4>Adenosylcobalamin (coenzyme B(12))-dependent enzymes catalyze a variety of chemically difficult reactions that proceed through the generation of free radical intermediates. A long-standing question is how proteins stabilize what are normally regarded as highly reactive organic radicals and direct them towards productive reactions. In glutamate mutase the carboxylate of Glu171 hydrogen bonds with the amino group of the substrate. We have investigated the role of this residue in the enzyme mechanism.<h4>Results</h4>Several sterically and functionally conservative mutations were introduced at position 171. In the most impaired mutant, Glu171Gln, k(cat) is reduced 50-fold, although the K(m) for glutamate is little affected. In the wild-type enzyme activity was pH-dependent and the acidic limb of the activity curve titrated with an apparent pK(a) of 6.6 on V(max), whereas for the sluggish Glu171Gln mutant activity is independent of pH. The steady state deuterium kinetic isotope effect is reduced in the mutant enzyme, but the steady state concentration of free radical species on the enzyme (as measured by the steady state concentration of cob(II)alamin) is unaffected by the mutation.<h4>Conclusions</h4>The properties of the mutant proteins are consistent with the hypothesis that Glu171 acts as a general base that serves to deprotonate the amino group of the substrate during catalysis. Deprotonation is expected to facilitate the formation of the glycyl radical intermediate formed during the inter-conversion of substrate and product radicals, but to have little effect on the stability of product or substrate radicals themselves. << Less

  Chem Biol 8:1143-1149(2001) [PubMed] [EuropePMC]