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Anaerobic bacteria ferment carbohydrates and amino acids to obtain energy for growth. Due to the absence of oxygen and other inorganic electron acceptors, the substrate of a fermentation has to serve as electron donor as well as acceptor, which results in low free energies as compared to that of aerobic oxidations. Until about 10 years ago, anaerobes were thought to exclusively use substrate level phosphorylation (SLP), by which only part of the available energy could be conserved. Therefore, anaerobes were regarded as unproductive and inefficient energy conservers. The discovery of electrochemical Na+ gradients generated by biotin-dependent decarboxylations or by reduction of NAD+ with ferredoxin changed this view. Reduced ferredoxin is provided by oxidative decarboxylation of 2-oxoacids and the recently discovered flavin based electron bifurcation (FBEB). In this review, the two different fermentation pathways of glutamate to ammonia, CO2, acetate, butyrate and H2 via 3-methylaspartate or via 2-hydroxyglutarate by members of the Firmicutes are discussed as prototypical examples in which all processes characteristic for fermentations occur. Though the fermentations proceed on two entirely different pathways, the maximum theoretical amount of ATP is conserved in each pathway. The occurrence of the 3-methylaspartate pathway in clostridia from soil and the 2-hydroxyglutarate pathway in the human microbiome of the large intestine is traced back to the oxygen-sensitivity of the radical enzymes. The coenzyme B12-dependent glutamate mutase in the 3-methylaspartate pathway tolerates oxygen, whereas 2-hydroxyglutaryl-CoA dehydratase is extremely oxygen-sensitive and can only survive in the gut, where the combustion of butyrate produced by the microbiome consumes the oxygen and provides a strict anaerobic environment. Examples of coenzyme B12-dependent eliminases are given, which in the gut are replaced by simpler extremely oxygen sensitive glycyl radical enzymes.

Coenzyme B12-dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B12-dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering.

Cobalamins are of widespread importance in biology. Both of the cofactors essential for human metabolism, the organocobalamins coenzyme B12 and methylcobalamin, are highly photolabile, as are other alkylcobalamins. The alkynylcobalamin phenylethynylcobalamin (PhEtyCbl) and the arylcobalamin 4-ethylphenylcobalamin (EtPhCbl) with “atypical” Co–C-bonds to unsaturated carbons, were recently designed as metabolically inert cobalamins, classified as “antivitamins B12”. The further development of an ideal light-activated or “conditional” antivitamin B12 would require it to be readily converted by light into an active B12 vitamin form. Very photolabile “antivitamins B12” would also represent particularly useful scaffolds for therapeutic light-activated reagents. Here, the photoactive arylcobalamin EtPhCbl and the remarkably photostable alkynylcobalamin PhEtyCbl are examined using femtosecond to picosecond UV–visible transient absorption spectroscopy. PhEtyCbl undergoes internal conversion to the ground state with...

High-energy radical species are widespread in biology as transient reaction intermediates and free radicals. The control of these extremely reactive molecules is crucial to limit oxidative stress and promote healthy physiological function.[1] In some cases this reactivity can be harnessed by a system to do useful work.[2] Over two decades ago Retey discussed how energy can be “borrowed” from an enzyme to effect an unlikely chemical change.[3] In the case of coenzyme B12-dependent enzymes,[4] this energy is “lent” in the form of radical intermediates, which are formed upon substrate binding by Co–C bond homolysis in the cofactor. The energy is then returned by radical pair recombination after turnover (Scheme 1).

The classical picture of enzyme catalysis relies on controlling the entropic and enthalpic contributions by manipulating reaction barriers and co-locating reactants and cofactors to facilitate the reaction chemistry. Catalysis is linked inextricably to the geometry of the enzyme–substrate complex and the chemical/physical properties of the active site, and probably to dynamical contributions that guide reactants along the desired reaction coordinate. Coenzyme B12-dependent enzymes have remarkable catalytic power and unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. Here we discuss recent developments that are beginning to provide atomistic insight into how coenzyme B12-dependent enzymes steer reactants along the reaction coordinate. Such insight will ultimately generate ‘movies’ of the catalytic process across all relevant time scales. In the longer term, this will enable more predictive engineering of this class of enzyme to achieve new and desirable chemical outcomes.

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

Adenosylcobalamin (AdoCbl), or coenzyme B12, is a cofactor for enzymes important in metabolism in humans (and other mammals) and bacteria. AdoCbl contains a Co-C bond and is extremely light sensitive, but, until recently, this light sensitivity appeared to have no physiological function. Recently, AdoCbl has been found to act as cofactor for a photoreceptor protein (CarH) that controls the expression of DNA coding for transcription of the proteins needed for synthesis of carotenes in certain non-photosynthetic bacteria. In 2015, the X-ray crystal structures of two dark states of the photoreceptor protein from the bacterium Thermus thermophilus were determined: CarH bound to AdoCbl and CarH bound to a large portion of the cognate DNA operator (and AdoCbl); a light state was also determined in which CarH was bound to cobalamin in which the Co-C bond had been broken. The breaking of the Co-C bond of Ado-Cbl acts as a trigger for the regulatory switch that allows the transcription of DNA. In the two dark states AdoCbl is bound to a conserved histidine from CarH, which displaces the lower 5,6-dimethylbenzimidazole ligand of AdoCbl. In the light state the 5’-deoxyadenosyl group of AdoCbl is replaced by a second histidine from CarH, giving a bis-histidine cobalamin and 4’,5’-anhydroadenosine. Genes for B12-dependent photoreceptors are widespread in bacteria. Control of DNA transcription may represent an evolutionarily ancient function of AdoCbl, possibly pre-dating its function as a protein cofactor. Significance: A new function for adenosylcobalamin, a light-sensitive form of vitamin B12 with a Co-C bond, has been discovered in bacteria Some non-photosynthetic bacteria use adenosylcobalamin as a cofactor for the protein CarH, which controls DNA transcription Three X-ray crystal structures of CarH have been determined: bound to adenosylcobalamin, DNA and after light exposure A mechanism of action for CarH, based on its structure and on model reactions of vitamin B12, is proposed

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B(12) or 5'-deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5'-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an approximately 3.5-fold lower K(act) for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.

Adenosylcobalamin (AdoCbl, coenzyme B12) is one of Nature's most complex enzyme cofactors and enables difficult radical chemistry via cleavage of its covalent cobalt-carbon bond. In a recently disc...

In the early days, radical enzyme reactions that use S-adenosylmethionine (SAM) coordinated to an Fe-S cluster, which Perry Frey described as a "poor man's coenzyme B12," were believed to be relatively rare chemical curiosities. Today, bioinformatics analyses have revealed the wide prevalence and sheer numbers of radical SAM enzymes, conferring superfamily status. In this thematic minireview series, the JBC presents six articles on radical SAM enzymes that accomplish wide-ranging chemical transformations. We learn that despite the diversity of the reactions catalyzed, family members share some common structural and mechanistic themes. Still in its infancy, continued explorations promise to be fertile grounds for discoveries that will undoubtedly further broaden our understanding of the catalytic repertoire and deepen our understanding of the chemical strategies used by radical SAM enzymes.

The reactivity and relative rarity of most cofactors pose challenges for their delivery to target enzymes. Using kinetic analyses, we demonstrate that adenosyltransferase, which catalyzes the final step in the assimilation of coenzyme B12, directly transfers the cofactor to methylmalonyl coenzyme A mutase. The strategy of using the final enzyme in an assimilation pathway for tailoring a cofactor and delivering it to a dependent enzyme may be general for cofactor trafficking.

In 1997 and the first half of 1998, numerous publications appeared reporting studies of cofactors and their analogues in classical model systems and in enzyme-catalyzed reactions directed at understanding the enzymatic reactions of their natural cofactors. Model systems based on flavins have provided new insights into enzymatic modulation of the flavin reduction potential, and enzymatic reactions of coenzyme A analogues and derivatives have been employed in several studies of coenzyme A utilizing enzymes. Coenzyme B12 analogues have been utilized as alternate cofactors for B12-utilizing enzymes, while pyrroloquinoline quinone esters and analogues have been employed in model studies of the reactions of quinoprotein-catalyzed reactions.

Thermodynamic and equilibria properties of two vitamin B12 coenzyme analogues, i.e. 1-methyl-5-deoxy-β-d-(−)ribofuranosylcobalamin (RibCbl) and 1-methyl-5-deoxy-2,3-isopropylidene-β-d-(−)ribofuranosylcobal- amin (Rib*Cbl) were studied by u.v.-vis. spectroscopy. Their temperature-dependent equilibria between 6-coordinate base-on species and 5-coordinate unprotonated base-off species, and pH-dependent equilibria for the protonation and displacement of 5,6-dimethylbenzimidazole at 25°C have been examined. Values of Δ H and Δ S in neutral solution and at pH 4.00, and the axial base pKa were determined. Comparing the results with those for coenzyme B12 and other cobalamins, it has been found that Δ H and Δ S values for two ribosylcobalamins fall in the range of organocobalamins, but for RibCbl*, a ca. 1 unit difference in pKa values exists. The effect of the ribofuranose structure on the Co-N(dbzm) strength is considered.

Three new analogs of coenzyme B12—2′,5′-dideosyadenssylcobinamide(2′dAdoCbi+), 2′,5′-dideoxycytidylcobinamide(2′dCytCbi+), and 5′-deoxythymidylcobinamide(ThyCbi+—were synthesized, at first, from tosylated 2′-deoxynucleosides and B12a to produce 2′,5′-dideoxynucleosidylcobalamins, and the to remove the DMBz nucleotide loop at pH = 8–9 using Ce(OH)3. They were characterized by UV-Vis and 1H NMR spectroscopies. A discussion on the spectral properties of them and a comparison with those of cobalamins containing 2′-deoxynucleosides were performed.

The ability of enzymes to harness free-radical chemistry allows for some of the most amazing transformations in nature, including reduction of ribonucleotides and carbon skeleton rearrangements. Enzyme cofactors involved in this chemistry can be large and complex, such as adenosylcobalamin (coenzyme B12), simpler, such as S-adenosylmethionine and an iron-sulfur cluster (i.e., poor man's B12), or very small, such as one nonheme iron atom coordinated by protein ligands. Although the chemistry catalyzed by these enzyme-bound cofactors is unparalleled, it does come at a price. The enzyme must be able to control these radical reactions, preventing unwanted chemistry and protecting the enzyme active site from damage. Here, we consider a set of radical folds: the (β/α)8or TIM barrel, combined with a Rossmann domain for coenzyme B12-dependent chemistry. Using specific enzyme examples, we consider how nature employs the common TIM barrel fold and its Rossmann domain partner for radical-based chemistry.

The 1HNMR spectrum of 2′,5′-dideoxyadenosylcobalamin, a Coenzyme B12(5′-deoxyadenosylcobalamin) analogue, has been assigned by 2D COSY. Its proton coupling constants have also been measured by J-resolved experiment. The comparison between the analogue and Coenzyme B12 was made.

The research in our laboratory focuses on the role of metal ions and their complexes in structure formation and folding of nucleic acids. Large catalytic RNAs, like group II introns and some riboswitches, as well as shorter RNAs and DNAs containing modified nucleotides for the assembly of nanodevices are examined. Abundant metal ions like Mg2+ or natural metabolites like coenzyme B12 are in the center of interest, but also other metal ions, complexes thereof and B12 derivatives are applied with the aim to understand the largely unknown and manifold non-covalent interactions with nucleic acids. We apply a multitude of techniques, including potentiometric pH titrations, NMR spectroscopy, X-ray crystallography, gel electrophoresis and single molecule FRET experiments. Here we briefly summarize each of our research topics emphasizing the interaction of coenzyme B12 and its derivatives with the btuB riboswitch of E. coli. This highly conserved sequence, found in the 5'-untranslated region (5'-UTR) of the btuB mRNA, is involved in the regulation of the btuB protein expression. After a summary on the historical discovery of such riboswitches and their mechanism of action, we shortly focus on our own contributions to understand the structural equilibrium, high affinity and selectivity of the interaction between this specific RNA sequence and the largest and most complex cellular metabolite, coenzyme B12.

Cobalamins are complex organometallic cofactors essential for catalysis in three enzyme families, the isomerases, methyltransferases and reductive dehalogenases. This account focuses on the isomerases, which catalyze difficult and unusual 1,2-rearrangement reactions. These enzymes utilize AdoCbl as a radical reservoir. They induce homolytic cleavage of the Co-C bond of the cofactor thus generating a highly reactive 5′-deoxyadenosyl radical that initiates the radical rearrangement process. Over the past decade, structure-function and computational studies of several members of the isomerase family have provided interesting insights into the mechanism of AdoCbl-dependent radical isomerizations. In particular, these studies have revealed the critical role played by the protein in catalyzing the trillion-fold rate enhancement of Co-C bond homolysis, in shielding radical intermediates from adventitious side-reactions and in facilitating the rearrangement process itself.

Many organic cofactors are both rare and reactive. They are usually in low abundance, which poses problems for efficient collision-based targeting to dependent enzymes, whereas their reactivity is problematic for side reactions. Sequestration and escorted delivery presents one solution to this conundrum, but such porters, if they exist, are mostly unknown. In humans, the mitochondrial enzyme methylmalonyl-coenzyme A mutase uses coenzyme B 12 (adenosylcobalamin) but would be inactive if bound to the cofactor precursor that is delivered to the mitochondrion. Adenosyltransferase converts cob(II)alamin to coenzyme B 12 . Based on kinetic evidence for interaction between the two enzymes, the 40-fold greater affinity for coenzyme B 12 and the higher coordination number for cobalt in the mutase, we propose that the adenosyltransferase is a dual-function protein: an enzyme that synthesizes coenzyme B 12 and a chaperone that delivers it.

Ab initio molecular orbital calculations are used to examine the mechanism of action of B(12)-dependent ethanolamine ammonia-lyase involving the conversion of 2-aminoethanol to acetaldehyde plus ammonia. We attempt to elucidate the mechanism by which the enzyme facilitates this reaction through interactions between active-site residues and the substrate. Our calculations suggest a preferred pathway involving a 1,2-shift in the associated radical and also suggest that interactions between the enzyme and the migrating group of the substrate that afford an almost fully protonated migrating group will lead to the most efficient catalysis. However, this criterion on its own is insufficient to fully understand the rearrangement. Additional synergistic interactions between the spectator hydroxyl group in the substrate and active-site residues on the enzyme are required to lower the barrier height to a value consistent with experimental observations.

Background: 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. Results: 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 p K 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. Conclusions: 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.

Adenosylcobalamin (coenzyme B12)-dependent glutamate mutase catalyzes a most unusual carbon skeleton rearrangement involving the isomerization of l-glutamate to L-threo-methylaspartate, a reaction that is without precedent in organic chemistry. This reaction proceeds through a mechanism involving free radical intermediates that are initiated by homolysis of the cobalt-carbon bond of the coenzyme. The enzyme serves as a paradigm for adenosylcobalamin-dependent catalysis and, more generally, provides insights into how enzymes generate and control reactive free radical species. This review describes how recent studies on the mechanism and structure of glutamate mutase have contributed to our understanding of adenosylcobalamin-mediated catalysis. Copyright 2000 Academic Press.

Highly active and cobamide-free glutamate mutase was obtained from Clostridium cochlearium by a modification of the original purification procedure. After incubation of the enzyme with dithiothreitol, adenosylcobalamin (coenzyme B12) and the substrate (S)-glutamate, a paramagnetic species was observed by EPR-spectroscopy. The signal was maximal within 15 ms after mixing with glutamate. Different signals were detected after incubating the system with the competitive inhibitors (2S,4S)-4-fluoroglutamate or 2-methyleneglutarate instead of the substrate. The former developed with an at least 100-fold lower rate then the signal with glutamate. All three signals are probably due to low-spin cob(II)amide species with an extraordinary low gxy value as compared with cob(II)alamin.

Partially purified 2-methyleneglutarate mutase from Clostridium barkeri was separated from 3-methylitaconate delta-isomerase by treatment with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) followed by FPLC on the anion exchange column Mono Q in the presence of the detergent. When purified in the dark, the active mutase contained a corrinoid, most probably coenzyme B12. The enzyme was inactivated by light at the same wavelength (lambda less than 620 nm) and rate as free coenzyme B12. The rate was not influenced by oxygen or by temperature (0-37 degrees C). Reactivation of up to 50% of the original activity was achieved by incubation with coenzyme B12 and dithiothreitol. The substrates 2-methyleneglutarate (up to 40 mM) or (R)-3-methylitaconate specifically protected the enzyme from inactivation by visible light. This effect was enhanced 3-fold by raising the temperature from 0 degrees C to 37 degrees C. The data indicate that during catalysis, the Co-C bond of the coenzyme is cleaved and cannot be affected any more by light.

Background: Glutamate mutase is an adenosylcobamide (coenzyme B 12 ) dependent enzyme that catalyzes the reversible rearrangement of (2 S )-glutamate to (2 S ,3 S )-3-methylaspartate. The enzyme from Clostridium tetanomorphum comprises two subunits (of 53.7 and 14.8 kDa) and in its active form appears to be an α 2 β 2 tetramer. The smaller subunit, termed MutS, has been characterized as the B 12 -binding component. Knowledge on the structure of a B 12 -binding apoenzyme does not exist. Results: The solution structure and important dynamical aspects of MutS have been determined from a heteronuclear NMR study. The global fold of MutS in solution resembles that determined by X-ray crystallography for the B 12 -binding domains of Escherichia coli methionine synthase and Propionibacterium shermanii methylmalonyl CoA mutase. In these two proteins a histidine residue displaces the endogenous cobalt-coordinating ligand of the B 12 cofactor. In MutS, however, the segment of the protein containing the conserved histidine residue forms part of an unstructured and mobile extended loop. Conclusions: A comparison of the crystal structures of two B 12 -binding domains, with bound B 12 cofactor, and the solution structure of the apoprotein MutS has helped to clarify the mechanism of B 12 binding. The major part of MutS is preorganized for B 12 binding, but the B 12 -binding site itself is only partially formed. Upon binding B 12 , important elements of the binding site appear to become structured, including an α helix that forms one side of the cleft accommodating the nucleotide ‘tail' of the cofactor.

Background: Methylmalonyl CoA mutase catalyses the interconversion of succinyl CoA and methylmalonyl CoA via a free radical mechanism. The enzyme belongs to a family of enzymes that catalyse intramolecular rearrangement reactions in which a group and a hydrogen atom on adjacent carbons are exchanged. These enzymes use the cofactor adenosylcobalamin (coenzyme B 12 ) which breaks to form an adenosyl radical, thus initiating the reaction. Determination of the structure of substrate-free methylmalonyl CoA mutase was initiated to provide further insight into the mechanism of radical formation. Results: We report here two structures of methylmalonyl CoA mutase from Propionibacterium shermanii . The first structure is of the enzyme in a nonproductive complex with CoA at 2.5 a resolution. This structure serves as a model for the substrate-free conformation of the enzyme, as it is very similar to the second much poorer 2.7 a resolution structure derived from a truly substrate-free crystal. The true substrate-free structure also shows the adenosyl group bound to the cobalt atom. Comparison of this structure with that of the previously reported complex of the enzyme with a substrate analogue shows that major conformational changes occur upon substrate binding. The substrate-binding site of the enzyme is located within a ( β α ) 8 TIM-barrel domain. In the absence of substrate, this TIM-barrel domain is split apart and the active site is accessible to solvent. When substrate binds, the barrel closes up with the substrate along its axis and the active site becomes completely buried. Conclusions: The closure of the active-site cavity upon substrate binding displaces the adenosyl group of the cofactor from the central cobalt atom into the active-site cavity. This triggers the formation of the free radical that initiates the rearrangement reaction. The TIM-barrel domain is substantially different from all others yet reported: in its unliganded form it is broken open, exposing the small hydrophilic sidechains which fill the centre. The typical barrel structure is only formed when substrate is bound.

Dehydratases have been detected in anaerobic bacteria which use 2-, 4- or 5-hydroxyacyl-CoA as substrates and are involved in the removal of hydrogen atoms from the unactivated beta- or gamma-positions. In addition there are bacterial dehydratases acting on 1,2-diols which are substrates lacking any activating group. These enzymes contain either FAD, or flavins + iron-sulfur clusters or coenzyme B12. It has been proposed that the overall dehydrations are actually reductions followed by oxidations or vice versa mediated by these prosthetic groups. Whereas the gamma-hydrogen of 5-hydroxyvaleryl-CoA is activated by a transient two-election alpha, beta-oxidation, the other substrates are proposed to require either a transient one-electron reduction or an oxidation to a ketyl (radical anion).

Background: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an α β heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B 12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA. Results Reported here is the crystal structure at 2 a resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B 12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B 12 -binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a ( β / α ) 8 TIM barrel domain. Conclusion The histidine–cobalt distance is very long (2.5 a compared with 1.95–2.2 a in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal–carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain.

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co-atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the gz-lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co-atom. A mixture of completely 15N-labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper gz-lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine-splitting of the gz-line, whereas the 15N-labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co-atom. By using a sample of 15N-labelled component S, in which only the histidines were 14N-labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.

Coenzyme B12 (5′-deoxyadenosylcobalamin, abbreviated [CoIIIR−]) serves as a cofactor for several enzymic reactions, a common feature of which involves the 1,2-interchange of a H atom and another group (X = OH, NH2, CH(NH2)COOH, etc.) on adjacent carbon atoms, i.e., A widely accepted mechanistic interpretation of these reactions, supported by a variety of evidence from studies on the enzymic processes as well as on model systems, is, At the present stage it appears that the principal, if not the only, role of coenzyme B12 is to serve as the precursor for an organic free radical which triggers the substrate rearrangement. This role implies and utilizes a very weak Coalkyl bond, susceptible to homolytic cleavage under mild conditions, for which there has until recently been no convincing evidence or precedent. We have developed the first reliable systematic methods for the determination of transition metalalkyl bond dissociation energies (BDE) and have applied these to the determination of such BDE's in a variety of organocobalt compounds (notably containing dioxime or Schiff base ligands) related to coenzyme B12. The CoC BDE's yielded by such measurements are in the range 15–25 kcal/mol, i.e., low enough to be consistent with the proposed role of coenzyme B12 depicted in Eqn. (1). The influences of electronic and steric factors on CoC bond dissociation energies, revealed by these studies, are such as to suggest that coenzyme B12 is an ideal candidate for such a role and that steric factors probably are responsible for the enzyme-induced CoC bond weakening and dissociation. An analogy is developed between the role of coenzyme B12 in biological systems as a reversible ‘free radical carrier’ (i.e., according to Eqn. (1)) and the role of hemes as reversible dioxygen carriers, i.e., [FeIIIO2−] ⇄ [FeII] + O2

Simple corrins such as vitamin B12 and vitamin B12 coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B12 coenzymes in enzymatic reactions.

Chloro-pyridino-bis (glyoximato)-cobalt(III) complexes (cobaloximes) have been synthesized in which two corners are connected by a bridge consisting of 10 or 12 methylene groups. These compounds are designed to mimic some important features of the active site of coenzyme B12 dependent enzymes.

The identity of the major cobamide in liver has not been satisfactorily established. Cyanocobalamin (vitamin B12, 5,6dimethylbenzimidazolylcobamide cyanide), which has been obtained in crystalline form with liver as a source material (1, 2), is certainly a very minor component of the cobalamins in liver. Analyses of liver concentrates, both by a paper chromatographicbioautographic method (3) and by a chemical method (4), have shown that cyanocobalamin is usually not present in detectable amounts and seldom constitutes as much as 10% of the total cobalamin. The preparation of cyanocobalamin from liver probably was made possible by the presence of cyanide in the charcoal used in the isolation process. Examination of a liver extract by the chromatographic-bioautographic method (3) indicated that hydroxocobalamin (vitamin B12b, 5,6-dimethylbenzimidazolylcobamide hydroxide) was the most abundant cobalamin. This compound was also obtained in pure form from liver (1, 5). However, the recent discovery of the cobamide coenzymes in bacteria (6,7) and the demonstration that they constitute a large part of the total cobamides in these organisms (8,9) raised the possibility that coenzyme Bit (5,6-dimethylbenzimidazolylcobamide coenzyme, DMBC coenzyme, DBC coenzyme) may be a constituent of liver. Since coenzyme B12, like the adenylcobamide coenzyme (lo), is relatively unstable and is readily converted to hydroxocobalamin or cyanocobalamin by treatment with light or cyanide,’ respectively, the inadvertent decomposition of the coenzyme, if present in liver extracts, could account for previous observations on the occurrence of the two cobalamin vitamins. The present paper reports some observations on the occurrence and abundance of a cobamide coenzyme, which has been identified as coenzyme Blz, in livers of rabbit, chicken, sheep, and man. A brief report of the occurrence of coenzyme Blz in rabbit liver has already appeared (7).