Coenzyme B12-dependent Enzymes Research Articles

A Combined Density Functional Theory and Molecular Mechanics Study of the Relationship between the Structure of Coenzyme B12 and Its Binding to Methylmalonyl-CoA Mutase

A combined density functional theory (DFT) and molecular mechanics (MM) approach was applied to investigate the relationship between the structure of a free coenzyme B12, and bound to methylmalonyl-CoA mutase. It was found that, upon coenzyme binding to apoenzyme, the Co-C bond remains intact, while the C-Naxial bond becomes slightly elongated and labilized. The labilization of the Co-Naxial bond that takes place in coenzyme B12-dependent enzymes is most likely necessary for fine-tuning of the cobalt-nitrogen (axial base) distance. The controlling of this distance is important to inhibit abiological site reaction involving heterolysis of the Co-C bond but is not important for biologically relevant Co-C bond homolysis.

Substrate-induced conformational change of a coenzyme B12-dependent enzyme: crystal structure of the substrate-free form of diol dehydratase.

Substrate binding triggers catalytic radical formation through the cobalt-carbon bond homolysis in coenzyme B12-dependent enzymes. We have determined the crystal structure of the substrate-free form of Klebsiella oxytoca diol dehydratase*cyanocobalamin complex at 1.85 A resolution. The structure contains two units of the heterotrimer consisting of alpha, beta, and gamma subunits. As compared with the structure of its substrate-bound form, the beta subunits are tilted by approximately 3 degrees and cobalamin is also tilted so that pyrrole rings A and D are significantly lifted up toward the substrate-binding site, whereas pyrrole rings B and C are only slightly lifted up. The structure revealed that the potassium ion in the substrate-binding site of the substrate-free enzyme is also heptacoordinated; that is, two oxygen atoms of two water molecules coordinate to it instead of the substrate hydroxyls. A modeling study in which the structures of both the cobalamin moiety and the adenine ring of the coenzyme were superimposed onto those of the enzyme-bound cyanocobalamin and the adenine ring-binding pocket, respectively, demonstrated that the distortions of the Co-C bond in the substrate-free form are already marked but slightly smaller than those in the substrate-bound form. It was thus strongly suggested that the Co-C bond becomes largely activated (labilized) when the coenzyme binds to the apoenzyme even in the absence of substrate and undergoes homolysis through the substrate-induced conformational changes of the enzyme. Kinetic coupling of Co-C bond homolysis with hydrogen abstraction from the substrate shifts the equilibrium to dissociation.

Controlling the reactivity of radical intermediates by coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Adenosylcobalamin or coenzyme B(12)-dependent enzymes are members of the still relatively small group of radical enzymes and catalyse 1,2-rearrangement reactions. A member of this family is methylmalonyl-CoA mutase, which catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA and, unlike the others, is present in both bacteria and animals. Enzymes that catalyse some of the most chemically challenging reactions are the ones that tend to deploy radical chemistry. The use of radical intermediates in an active site lined with amino acid side chains that threaten to extinguish the reaction by presenting alternative groups for abstraction poses the conundrum of how the enzymes control their reactivity. In this review, insights into this issue that have emerged from kinetic, mutagenesis and structural studies are described for methylmalonyl-CoA mutase.

Radical Shuttling in a Protein: Ribose Pseudorotation Controls Alkyl-Radical Transfer in the Coenzyme B12 Dependent Enzyme Glutamate Mutase

Two alternate conformations of the adenosyl ligand of the cofactor are observed in the active site of the coenzyme B12 dependent enzyme glutamate mutase. This result shows ribose pseudorotation to be an elegant and safe mechanism for shuttling the “hot” methylene radical between the cofactor and substrate (see scheme).

Radical peregrinations catalyzed by coenzyme B12-dependent enzymes.

Radical peregrinations catalyzed by coenzyme B12-dependent enzymes.

Synthesis and Characterization of 3‘,4‘-Anhydroadenosylcobalamin: A Coenzyme B12 Analogue with Unusual Properties

The question of how coenzyme B12-dependent enzymes facilitate the cleavage of the Co−C bond of the cofactor is of interest. We have synthesized an analogue of 5‘-deoxyadenosylcobalamin (AdoCbl1) designed to stabilize the 5‘-deoxyadenosyl radical (5‘-deoxyadenosine-5‘-yl) that is produced upon homolysis of the Co−C bond. By replacement of the upper axial ligand of AdoCbl by a 3‘,4‘-anhydro-5‘-deoxyadenosyl moiety, the radical formed on the nucleoside analogue is stabilized by allylic delocalization. The compound, 5‘-deoxy- 3‘,4‘-anhydroadenosylcobalamin (3‘,4‘-anAdoCbl) was synthesized by chemical and enzymatic methods. The final step was coupling of cob(I)alamin and 3‘,4‘-anhydroATP catalyzed by CobA, an ATP:corrinoid adenosyltransferase. 3‘,4‘-anAdoCbl displays interesting properties. The compound has not been purified to homogeneity due to its thermal and oxygen sensitivity. It was characterized by UV−vis spectroscopy, ESI-MS, and NMR spectroscopy. The bond dissociation energy of the Co−C bond of the analogue was measured by radical trapping techniques. A significantly weaker bond (24 ± 2 kcal/mol) as compared to AdoCbl (30 kcal/mol) was observed, as was homolytic cleavage at ambient temperature. Photolysis experiments conducted under anaereobic conditions reveal no formation of cob(II)alamin, whereas the compound breaks down rapidly under aerobic conditions as measured by cob(III)alamin formation. We postulate that the weak Co−C bond is cleaved reversibly by photolysis, where recombination of the allylic radical and cob(II)alamin occurs efficiently in the absence of a radical scavanger. Activation of the coenzyme B12-dependent enzymes diol dehydrase and ethanolamine ammonia-lyase was observed with the cofactor analogue. The measured activity was low and no formation of cob(II)alamin could be detected in the steady-state of the reaction for either enzyme. Comparative interactions of AdoCbl and 3‘,4‘-anAdoCbl with diol dehydrase and ethanolamine ammonia-lyase suggest that cleavage of the Co−C bond is facilitated by enzyme-coenzyme binding contacts that are remote from the Co−C bond.

EXAFS Data Indicate a “Normal” Axial Cobalt−Nitrogen Bond of the Organo-B12 Cofactor in the Two Coenzyme B12-Dependent Enzymes Glutamate Mutase and 2-Methyleneglutarate Mutase

A key step in the catalytic cycle of coenzyme B12-dependent enzymes is the homolysis of the cofactor's organometallic bond, leading to the formation of a 5‘-deoxyadenosyl radical. For the adenosylcobalamin-dependent enzyme methylmalonyl CoA mutase (MCM), it has been suggested that this step is mediated by a protein-induced lengthening of the cofactor's axial cobalt−nitrogen bond, in trans position to the scissile organometallic bond. In fact, such a lengthening was first observed in the crystal structure of MCM (Mancia et al. Structure 1996, 4, 339−350) and was later confirmed by an analysis of EXAFS data on the same protein in frozen solution (Scheuring et al. J. Am. Chem. Soc. 1997, 119, 12192−12200). Here, we report the results of an EXAFS study on the related coenzyme B12-dependent enzymes glutamate mutase from Clostridium cochlearium and 2-methyleneglutarate mutase from Clostridium barkeri. Both apoenzymes were overproduced from E. coli and reconstituted with methylcobalamin (MeCbl) to yield inactive...

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

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. 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. 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.

Magnetic field effects on coenzyme B12-dependent enzymes: validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase.

Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B12-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5'-deoxyadenosyl radical and cob(II)alamin [Ado. Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase.

Calculations of Earth-strength steady and oscillating magnetic field effects in coenzyme B12 radical pair systems

This paper uses electron paramagnetic resonance data for radical pair systems in several coenzyme B12-dependent enzymes and calculates effects of Earth-strength steady and oscillating magnetic fields on their singlet-to-triplet yields via the radical pair mechanism. Energy level repulsions and the state mixing that they induce are found to be very important for determining overall sizes of effects and lower bounds on oscillating-field frequencies that can cause effects in such systems. B12 and similar systems with nearly axial zero-field spin Hamiltonians, dominated by terms over 100 times larger than Zeeman terms due to Earth-strength steady fields, if under relatively immobile conditions of long lifetime, slow molecular tumbling and slow spin relaxation, may be useful as biological sensors of both steady and oscillating fields that occur in nature, since the yields calculated show sensitivity to steady field strength (even after powder averaging) and orientation and undergo steady-field-dependent shifts...

Structural behaviour of cobaloximes: planarity, an anomalous trans-influence and possible implications on CoC bond cleavage in coenzyme-B 12-dependent enzymes

Deviations from planarity of the 17 non-hydrogen atoms forming the equatorial moiety in cobaloximes can be described by six significant distortion modes. No significant correlation has been found between these deformations and changes in the axial CoC and CoN distances. Analysis of the CoR and CoB distances reveals that, in a series of related RCo(DH) 2B complexes, the two distances lengthen or shorten simultaneously but to different degrees. This anomalous structural trans-influence is quantified with the help of an additive scheme based on the nature of the axial ligands R and B, and is discussed in terms of a simple Hückel molecular-orbital model.

Structural behaviour of cobaloximes: planarity, an anomalous trans-influence and possible implications on Co-C bond cleavage in coenzyme-B 12-dependent enzymes

Deviations from planarity of the 17 non-hydrogen atoms forming the equatorial moiety in cobaloximes can be described by six significant distortion modes. No significant correlation has been found between these deformations and changes in the axial Co-C and Co-N distances. Analysis of the Co-R and Co-B distances reveals that, in a series of related RCo(DH) 2B complexes, the two distances lengthen or shorten simultaneously but to different degrees. This anomalous structural trans-influence is quantified with the help of an additive scheme based on the nature of the axial ligands R and B, and is discussed in terms of a simple Hückel molecular-orbital model.

Glutamate and 2-methyleneglutarate mutase: from microbial curiosities to paradigms for coenzyme B12-dependent enzymes

Glutamate and 2-methyleneglutarate mutase: from microbial curiosities to paradigms for coenzyme B12-dependent enzymes

Intramolecularly alkylated cobalt complexes as models for the active site of coenzyme B12 dependent enzymes

Intramolecularly alkylated cobalt complexes as models for the active site of coenzyme B12 dependent enzymes

Accurate Structural Data Demystify B12: High-Resolution Solid-State Structure of Aquocobalamin Perchlorate and Structure Analysis of the Aquocobalamin Ion in Solution

Experiments are described to elucidate the structure and solvation of aquocobalamin (vitamin Biza, 1+) in the crystal and in aqueous solution. Aquocobalamin (1+) is the B12 derivative in which a water molecule replaces the axially coordinating organic substituent (methyl or 5'-desoxyadenosyl) at the P-side of the cobalt of the B12 coenzymes. (1) A single-crystal structure analysis of aquocobalamin perchlorate (l'ClO,), using synchrotron radiation in combination with an imaging plate detector, yielded the most accurate structural data ever determined for a B12 molecule. l'C10, crystallizes in the orthorhombic space group P212121, a = 15.042(11) A, b = 23.715(14) A, c = 25.104(12) A, with four 1'CIOi moieties plus about 100 solvent water molecules per unit cell; 22 867 independent and 20 942 significant intensity data were recorded to a nominal resolution of 0.8 A, and refinement against quantities led to a conventional R-value of 0.050 for all 22 867 observations and to a structural model with an average ESD for all carbon-carbon bonds of 0.003 A. In the crystal, the aquocobalamin ion has a very short axial bond between cobalt and the dimethylbenzimidazole (DMB) base of 1.925 (0.002) A, which is rationalized as resulting from the very weak trans axial donor (water). Steric repulsion between the DMB base and the corrin ring induced by this short Co-DMB bond leads to a relatively large butteffly with an fold angle of 18.7 l(0.07). The relevance of this observation for the upward conformational deformation hypothesis for the initiation of Co-C bond homolysis in coenzyme B12 dependent enzymes is discussed. (2) EXAFS spectra were taken from an authentic sample of the aquocobalamin perchlorate crystals used for the X-ray structure analysis, as well as from 1+C104 dissolved in a 1:l mixture of watedethylene glycol. Absorption spectra were recorded at 20 K between 7300 and 8700 eV, and the k3-weighted EXAFS was extracted for a k-value between 2.7 and 14.4 A-l. EXAFS spectra for solid and dissolved l'C10, agree closely, establishing identical cobalt coordination for the aquocobalamin ion in solution and in the solid state. A curve-fitting analysis on the Fourier filtered first-shell data yields a coordination number of 6 and an average distance of 1.90 A for both samples. There is no evidence for a longer Co-N distance. This refutes data published by Sagi and Chance (J. Am. Chem. Soc. 1992, 114, 8061). (3) NMR experiments are described, constituting the first detailed NMR investigations on a Bl2 derivative in H20, including 2D homo- and heteronuclear studies on aquocobalamin chloride (l+Cl-) and assignment of signals due to the exchangeable amide protons of all nitrogens as well a measurements of amide proton exchange rates. The NMR data confirm the occupation of the axial coordination site at the Co(II1) center by water, as well as the occurrence in solution of an intramolecular hydrogen bond to the axially coordinating water molecule, as observed in the crystal structure of l'C10,. However, significant differences of the structure of 1+ in crystals of l'C10, and of 1+ in aqueous solution are indicated from NOE data concerning the time-averaged conformation of the hydrogen-bonding c-acetamide side chain.

Magnetic field effects on B12 ethanolamine ammonia lyase: evidence for a radical mechanism.

A change in radical pair recombination rates is one of the few mechanisms by which a magnetic field can interact with a biological system. The kinetic parameter Vmax/Km (where Km is the Michaelis constant) for the coenzyme B12-dependent enzyme ethanolamine ammonia lyase was decreased 25 percent by a static magnetic field near 0.1 tesla (1000 gauss) with unlabeled ethanolamine and decreased 60 percent near 0.15 tesla with perdeuterated ethanolamine. This effect is likely caused by a magnetic field-induced change in intersystem crossing rates between the singlet and triplet spin states in the [cob(II)alamin:5'-deoxyadenosyl radical] spin-correlated radical pair.

Adocobalamin (AdoCbl or coenzyme B12) cobalt-carbon bond homolysis radical-cage effects: product, kinetic, mechanistic, and cage efficiency factor (Fc) studies, plus the possibility that coenzyme B12-dependent enzymes function as "ultimate radical cages" and "ultimate radical traps"

Following an introduction into solvent radical-cage effects and the general use of cage-trapping methods to study solvent-cage effects, the bio-organometallic enzyme cofactor coenzyme B 12 (adocobalamin; AdoCbl) in studied using the TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxy) nitroxide cage-trapping method. Specifically, the products, kinetics, and mechanism of AdoCbl Co-C thermolynin in ethylene glycol are presented at high [TEMPO], results which establish (after ruling out other conceivable mechanisms and interpretations) a radical-cage effect for AdoCbl in ethylene glycol

Organocobalamin Reactions Relevant to the Mechanism of the α-Methyleneglutarate Mutase Enzyme [1

Abstract The organocobalamin with methylitaconic acid attached to cobalt via its methyl group has been synthesized for the first time. This compound is believed to be an intermediate in the interconversion of methylitaconic acid and α-methyleneglutaric acid, catalysed by the coenzyme B12-dependent α-methyleneglutarate mutase enzyme. Reactions of this organocobalamin and the corresponding dimethyl ester demonstrate that cleavage of the Co-C bond leads to the rearranged α-methyleneglutarate structure under conditions where intermediate carbanions are formed.

Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases.

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source.

The mechanism of in sutu reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehydratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninylbutylcobalamin and adenosylethylcobalamin were essentially not displaceable by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Co beta ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are reactivated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism).