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Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F 430 , an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCR red1 ) contains Ni(I)-F 430 . When 3-bromopropane sulfonate (BPS) is incubated with MCR red1 , an alkyl-Ni(III) species is formed that elicits the MCR PS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRps species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCR red1 state. When reacted with 4-bromobutyrate, MCR red1 forms the alkyl-Ni(III) MCR XA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCR red1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.

Glycerol dehydratase (EC 4.2.1.30), as one of the key enzymes in converting glycerol to the valuable intermediate 1,3-propanediol, is important for biochemical industry. The dhaB genes encoding coenzyme B(12)-dependent glycerol dehydratase in Klebsiella pneumoniae were cloned and expressed in Escherichia coli. An effective co-expression system of multiple subunits protein was constructed. Heterologous expression vectors were constructed using the splicing by overlap extension-PCR technique to co-express the three subunits of the glycerol dehydratase. After induction by isopropyl-beta-D-thiogalactopyranoside, SDS-PAGE analysis revealed that: (i) only the alpha subunit of glycerol dehydratase was expressed in direct expression system, (ii) the three subunits of glycerol dehydratase with predicted molecular massess of 64 (agr;), 22 (beta), and 16 kDa (gamma) were expressed simultaneously in co-expression system, and (iii) the fusion expression system expressed the fusion protein of 99 kDa. Enzyme assay showed that the activities of three heterologous expression products were 27.4, 2.3, and 0.2 U/mg. The highest enzyme activity was almost 17 times of that in K. pneumoniae. The recombinant enzyme was purified and biochemically characterized. The apparent Km values of the enzyme for coenzyme B(12) and 1, 2-propanediol were 8.5 nM and 1.2 mM, respectively. The enzyme showed maximum activity at pH 8.5 and 37 degrees C.

Arginine 100 plays an important role in substrate recognition in adenosylcobalamin-dependent glutamate mutase. We have examined how the mutation of this residue to lysine affects the partitioning of tritium, incorporated at the exchangeable position of the coenzyme, between substrate and product. We find that partitioning of tritium back to the substrate predominates in the mutant enzyme, regardless of whether the reaction is run in the forward or reverse direction. This contrasts with the behavior of the wild-type enzyme in which tritium partitions equally between substrate and product, independent of the direction of the reaction. From this we conclude that the mutation significantly impairs the ability of the enzyme to catalyze the rearrangement of substrate radical to product radical. The results illustrate the importance of electrostatic interactions in stabilizing free radical intermediates in this class of enzymes.

Kinetic isotope effects provide a powerful method to investigate the mechanisms of enzyme-catalyzed reactions, but often other slow steps in the reaction such as substrate binding or product release suppress the isotopically sensitive step. For reactions at methyl groups, this limitation may be overcomed by measuring the isotope effect by an intra-molecular competition experiment. This requires the synthesis of substrates containing regio-specifically mono- or di-deuterated methyl groups. To facilitate the mechanistic investigations of the adenosylcobalamin-dependent enzyme, glutamate mutase, we have developed a synthesis of mono- and di-deuterated (2S,3S)-3-methylaspartic acids. Key intermediates are the correspondingly labeled mesaconic acids and their dimethyl esters that potentially provide starting materials for a variety of isotopically labeled molecules.

The “complete” corrinoid 176-norpseudovitamin B12 was recently isolated as the cyano-Co(III)-form of the corrinoid cofactor of tetrachlorethene reductive dehalogenase of the anaerobe Sulfurospirillum (formerly Dehalospirillum) multivorans. 176-Norpseudovitamin B12 represents the first example of (the cyano-Co(III)-form of) a naturally occurring “complete” B12 cofactor lacking a characteristic peripheral methyl group of the cobamide ligand. Its discovery has generated interest in 176-nor-B12 derivatives, “complete” corrinoids lacking the methyl group attached to carbon 176. Here, we report the preparation of Coβ-5′-adenosyl-176-norcobinamide by in situ alkylation of Co(I)-176-norcobinamide, obtained from electrochemical reduction of Coα,Coβ-dicyano-176-norcobinamide. Since Coβ-5′-adenosylcobinamide is a biosynthetic intermediate of the complete cobamides, Coβ-5′-adenosyl-176-norcobinamide is a “rational” biosynthetic precursor for natural 176-nor-B12 derivatives. The spectroscopic data for adenosyl-176-norcobinamide establish the suggested structure of the title compound and give further evidence for the extensive flexibility and conformational dynamics of the organometallic 5′-deoxy-5′-adenosyl ligand.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

Riboswitches are cis-regulatory RNA elements on the mRNA level that control the expression of the downstream coding region. The interaction of the riboswitch with its specific metabolite, which is related to the function of the controlled gene, induces a structural change of the RNA architecture. Consequently, gene regulation is induced by un/masking of the ribosome binding site (RBS). In the genome of Klebsiella pneumoniae a sequence was identified by bioinformatics and proposed to be a B12 riboswitch regulated by coenzyme B12. Here we study this new coenzyme B12-dependent riboswitch system by in-line probing and ITC. The riboswitch sequence includes the whole expression platform as well as RBS. In-line probing experiments were performed to investigate the structural rearrangement of this 243-nt long RNA sequence while Isothermal Titration Calorimetry (ITC) yielded the thermodynamic parameters of the interaction between the riboswitch and its metabolite. The interaction of coenzyme B12 with the butB riboswitch of K. pneumoniae is an exothermic process with a 1:1 binding stoichiometry and binding affinities of log KA = 6.73 ± 0.02 at 15 °C and log KA = 6.00 ± 0.09 at 30 °C.

Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.

Although methanogenic archaea use B 12 extensively as a methyl carrier for methanogenesis, little is known about B 12 metabolism in these prokaryotes or any other archaea. To improve our understanding of how B 12 metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobA Mm here) was cloned and used to restore coenzyme B 12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA Mm protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca 2+ ions, but the activity was not enhanced by Mg 2+ and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn 2+ . The CobA Mm enzyme had a K m ATP of 3 μM and a K m HOCbl of 1 μM. Unlike the S. enterica enzyme, CobA Mm used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the β phosphate of ATP was required for binding to the enzyme. A striking difference between CobA Se and CobA Mm was the use of ADP as a substrate by CobA Mm , suggesting an important role for the γ phosphate of ATP in binding. The results from 31 P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP i ) is the reaction by-product; no cleavage of PPP i was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP i ). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA Se and CobA Mm enzymes.

Crystal structures of a kind of cyclohexyl-cobaldioxime coenzyme B12 model compounds RCo(chgH)2L (R = CH3, C2H5 or C3H7, L = pyridine or H2O, chgH = 1,2-cyclohexanedione dioxime) at room and low temperatures have been determined. The results for C2H5Co(chgH)2py (1) indicate that its crystal unit parameters vary with temperatures, and two sides of cyclohexyls on the equatorial plane assume disorder accompanying a temperature dependent conformation occupation change. Interestingly, there has been observed a proton shift from one chgH ligand to the other one for 1 when the temperature decreases. Whilst a similar chair-form configuration with disordered chgH is found at 293 K for CH3Co(chgH)2py (2), C2H5Co(chgH)2H2O (3), and C3H7Co(chgH)2H2O (4), respectively. In addition, molecular and crystal packing structures, as well as thermal decomposition properties of the four complexes are investigated and compared with other cobaloxime models.

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR Ni(II)-*SR.

Despite decades of research, the mechanism by which coenzyme B 12 (adenosylcobalamin, AdoCbl)-dependent enzymes promote homolytic cleavage of the cofactor's Co-C bond to initiate catalysis has continued to elude researchers. In this work, we utilized magnetic circular dichroism spectroscopy to explore how the electronic structure of the reduced B 12 cofactor (i.e., the post-homolysis product Co 2+ Cbl) is modulated by the enzyme methylmalonyl-CoA mutase. Our data reveal a fairly uniform stabilization of the Co 3d orbitals relative to the corrin π/π*-based molecular orbitals when Co 2+ Cbl is bound to the enzyme active site, particularly in the presence of substrate. Contrastingly, our previous studies (Brooks, A. J.; Vlasie, M.; Banerjee, R.; Brunold, T. C. J. Am. Chem. Soc. 2004, 126, 8167-8180.) showed that when AdoCbl is bound to the MMCM active site, no enzymatic perturbation of the Co 3+ Cbl electronic structure occurs, even in the presence of substrate (analogues). Collectively, these observations provide direct evidence that enzymatic Co-C bond activation involves stabilization of the post-homolysis product, Co 2+ -Cbl, rather than destabilization of the Co 3+ Cbl ground state.

Density functional calculations have been used to compare the geometric, electronic, and functional properties of the three important tetrapyrrole systems in biology, heme, coenzyme B 12, and coenzyme F430, formed from iron porphyrin ( Por ), cobalt corrin ( Cor ), and nickel hydrocorphin ( Hcor ). The results show that the flexibility of the ring systems follows the trend Hcor > Cor > Por and that the size of the central cavity follows the trend Cor < Por < Hcor . Therefore, low-spin Co I, Co II, and Co III fit well into the Cor ring, whereas Por seems to be more ideal for the higher spin states of iron, and the cavity in Hcor is tailored for the larger Ni ion, especially in the high-spin Ni II state. This is confirmed by the thermodynamic stabilities of the various combinations of metals and ring systems. Reduction potentials indicate that the +I and +III states are less stable for Ni than for the other metal ions. Moreover, Ni – C bonds are appreciably less stable than Co - C bonds. However, it is still possible that a Ni – CH 3 bond is formed in F 430 by a heterolytic methyl transfer reaction, provided that the donor is appropriate, e.g. if coenzyme M is protonated. This can be facilitated by the adjacent SO 3− group in this coenzyme and by the axial glutamine ligand, which stabilizes the Ni III state. Our results also show that a Ni III– CH 3 complex is readily hydrolysed to form a methane molecule and that the Ni III hydrolysis product can oxidize coenzyme B and M to a heterodisulphide in the reaction mechanism of methyl coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH(3)-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an alpha(2)beta(2)gamma(2) subunit structure forming two structurally interlinked active sites each with a molecule F(430) as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV-vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV-vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20 degrees C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed.

Earlier calculations of the possible involvement of the axial base of coenzyme B12 in the mechanism of its enzymatic activation for Co–C bond homolysis led to the conclusion that classical ground state ‘mechanochemical triggering’, in which compression of the axial Co–N bond leads to destabilization of the Co–C bond in the ground state, was unlikely to provide significant catalytic power, but that so-called ‘transition state mechanochemical triggering’, in which such bond compression electronically stabilizes the transition state by improving the Co–N overlap, was a viable candidate for the mechanism of coenzyme activation. However, it is now clear that Co–C bond homolysis of coenzyme B12 at an enzyme active site is a simple bond dissociation reaction, the reaction coordinate diagram has no maximum, and there is no transition state. Consequently, the reaction can be accelerated only by destabilization of the reactant or stabilization of the homolysis products. In the current work, the possibility of stabilization of the cob(II)alamin product by compression of the axial Co–N bond to improve orbital overlap is addressed by molecular modeling and molecular orbital calculations. Two different semi-empirical open-shell calculations give differing results, so that the feasibility of such product stabilization cannot be answered unequivocally by such calculations.

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F(430) as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K(M)=20 mM; apparent V(max)=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K(M)=5 mM; apparent V(max)=30 U/mg). Propyl-coenzyme M (apparent K(i)=2 mM) and allyl-coenzyme M (apparent K(i)=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I](0.5 V)=2 micro M), cyano-coenzyme M ([I](0.5 V)=0.2 mM), 3-bromopropionate ([I](0.5 V)=3 mM), seleno-coenzyme M ([I](0.5 V)=6 mM) and trifluoromethyl-coenzyme M ([I](0.5 V)=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D(2)O. The signal was abolished upon exposure of the enzyme to O(2). 3-Bromopropanesulfonate ([I](0.5 V)=0.1 micro M), 3-iodopropanesulfonate ([I](0.5 V)=1 micro M), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O(2). 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.

The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself. NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data. Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand. Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid. Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways. A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways. Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature. At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself. It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are similar to those of AdoCbl itself, and that this is responsible for the high enzymatic activity of this analog.

The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B(12) from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 A resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa alpha subunits and two globular 14 kDa beta subunits. The alpha subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the beta subunit resembles that of the beta subunit of glycerol dehydratase, except that it lacks some coenzyme B(12) binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl-methanofuran synthesis is an endergonic reaction (ΔG°′ = +16 kJ·mol−1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (ΔG′) were found to range between +10 and +35 kJ·mol−1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (ΔG°′ = −40 kJ·mol−1) was associated with free energy changes as high as −55 to −80 kJ·mol−1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2

Coenzyme B 12 is an organometallic compound that catalyzes biological rearrangement reactions. Homolytic cleavage of the unusual cobalt-carbon bond in the coenzyme initiates the free-radical reaction. In an attempt to understand the factors that might be important in the mechanism, we synthesized a series of model complexes [LCo{(DO)(DOH)bn}R] + with a folded equatorial ligand and determined the crystal structures. Semi-empirical calculations with these and other model compounds provide evidence for a transelectronic influence; when the Co-N bond is shortened, the Co-C bond lengthens. These results are compared to density functional calculations carried out by others.

A peculiar function resides in a peculiar structure. Coenzyme B(12) or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three-dimensional structures of several radical-containing or radical-forming enzymes including B(12) enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin-dependent enzyme, revealed that its structure is adapted for its function-that is, activation of the Cobond;C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate-derived radical. The functions of K(+) and the active-site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2β2γ2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called ‘base-on’ mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.

The design and implementation of a new, higher yield synthetic method for synthesizing zwitterionic cobinamide phosphates is described. Adenosylcobinamide 2-chlorophenyl phosphate, beta-AdoCbi-PAr -- a 5,6-dimethylbenzimidazole-free adenosylcobalamin analog, where a 2-chlorophenyl group replaces the ribofuranose and 5,6-dimethylbenzimidazole moieties -- is prepared in tens of milligram quantities, quantities sufficient for crystallization and enzyme trials, amounts 100-fold greater than previously available. The use of (31)P NMR spectroscopy to follow reactions directly, the use of control reactions to learn how to reduce reactant water content, and the use of reaction solvents that completely dissolved the corrinoid reactants were crucial for developing this new synthetic route. beta-AdoCbi-PAr was synthesized in 10% overall isolated yield from cyanocobinamide. Cyanocobinamide was converted to cyanocobinamide 2-chlorophenyl phosphate by direct phosphorylation with 2-chlorophenyl phosphodi-(1,2,4-triazolide) in 25% isolated yield andor = 98% purity. Sodium borohydride reduction of cyanocobinamide 2-chlorophenyl phosphate and reaction with 5'-chloro-5'-deoxy-adenosine produced beta-AdoCbi-PAr in 42% yield andor = 98% purity. These compounds were characterized by HPLC, (1)H and (31)P NMR, UV-visible spectroscopy, and liquid secondary ionization mass spectroscopy.

The compound 8-methoxy-5'-deoxyadenosylcobalamin (8-MeOAdoCbl), has been synthesized in 37% yield andor = 95% purity by HPLC, monitored at both 254 and 525 nm, or 90+/-2% purity as judged by the (1)H NMR spectrum of the aromatic cobalamin region. This is the first synthesis of this complex in which sufficient details are reported, where a yield and purity are reported, and where key problems in the synthesis and purification are overcome, so that 8-MeOAdoCbl can actually be obtained for use in other studies. Also demonstrated is the clean Co-C bond homolysis of 8-MeOAdoCbl to give initially 8-MeOAdoCbl and Co(II)Cbl in a UV-visible thermolysis experiment at 110 degrees C, results which show that the 8-MeO moiety suppresses the cyclization to the 8,5'-anhydro-adenosine otherwise seen for the adenosyl radical (Ado)*. Suppression of this cyclization pathway makes 8-MeOAdoCbl invaluable for studying the kinetic isotope effect (KIE) of the Ado* plus substrate H* abstraction reaction, a component of the first definitive test of Klinman's hypothesis that the optimization of enzyme catalysis may entail strategies that increase the probability of tunneling and thereby accelerate H* atom abstraction reaction rates.

A novel bridged organocobaloxime-bromo(O-trimethylene-CHEL)cobaloxime was synthesized by heating the Aqua(3-bromopropyl)cobaloxime at 50 °C in methanol/H2O and crystal structure was determined by X-ray diffraction analysis.

Structural Biology Most of the methane on Earth is produced by the metabolism of methanogenic archaea. The final step involves a reaction between methyl-coenzyme M and coenzyme B to give CoM-S-S-CoB and methane. Wagner et al. report a high-resolution structure of the methanogenic heterodisulfide reductase (HdtABC)-[NiFe]-hydrogenase, the enzyme that reduces the disulfide and couples this to the reduction of ferredoxin in an energy-conserving process known as flavin-based electron bifurcation (FBEB) (see the Perspective by Dobbek). The reduced ferredoxin, in turn, drives the first step of methanogenesis. The structure shows how two noncubane [4Fe-4S] clusters perform disulfide cleavage and gives insight into the mechanism of FBEB. Science , this issue p. [699][1]; see also p. [642][2] [1]: /lookup/doi/10.1126/science.aan0425 [2]: /lookup/doi/10.1126/science.aao2439

Crystal structure analyses have helped to decipher the mode of binding of coenzyme B12 (AdoCbl) in the active site of AdoCbl-dependent enzymes. However, the question of how such enzymes perform their radical reactions is still incompletely answered. A pioneering study by Gruber and Kratky of AdoCbl-dependent glutamate mutase (GLM) laid out a path for the movement of the catalytically active 5′-deoxyadenosyl radical, in which H-bonds between the protein and the 2′- and 3′-OH groups of the protein bound AdoCbl would play a decisive role. Studies with correspondingly modified coenzyme B12-analogues are of interest to gain insights into cofactor binding and enzyme mechanism. Here we report the preparation of Coβ-2′-fluoro-2′,5′-dideoxyadenosylcobalamin (2′FAdoCbl), which lacks the 2′-OH group critical for the interaction in enzymes. 2′FAdoCbl was prepared by alkylation of cob(I)alamin, obtained from the electrochemical reduction of aquocobalamin. Spectroscopic data and a single crystal X-ray analysis of 2′FAdoCbl established its structure, which was very similar to that one of coenzyme B12. 2′FAdoCbl is a 19F NMR active mimic of coenzyme B12 that may help to gain insights into binding interactions of coenzyme B12 with AdoCbl-dependent enzymes, proteins of B12 transport and of AdoCbl-biosynthesis, as well as with B12-riboswitches.

Recent progress in computational modeling of the catalytic activation of cobalt-carbon bond cleavage shows that quantum chemical calculations could be an important part of coenzyme B(12) research. Particular emphasis has been placed on density functional theory, which is now emerging as a powerful tool to elucidate the electronic structure and spectroscopic properties of the active sites of metalloenzymes.

Coenzyme B(12) serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B(12)-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G* level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1,2-propanediol as substrate are 9.0 and 15.1 kcal/mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B(12)-dependent diol dehydratase reaction are energetically feasible.

Uniformly (13)C,(15)N-labeled MutS, the coenzyme B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of (13)C,(15)N-labeled MutS provided signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with (15)N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B(12)-binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B(12)-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an alpha-helical structure ("nascent helix"). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B(12) and appears to be stabilized as a helix (alpha1) upon trapping of the nucleotide of the B(12) cofactor.

The biochemical route for the formation of the phosphodiester bond in coenzyme F(420), one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F(420) structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactate represents a new natural product that was chemically identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc. jannaschii. Incubation of cell extracts of both M. thermophila and Mc. jannaschii with [hydroxy-(18)O, carboxyl-(18)O(2)]lactate and GTP produced 2-phospho-L-lactate with the same (18)O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex G-25 purified cell extracts of M. thermophila or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F(420)-0 (F(420) with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F(420)-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extracts with L-[U-(14)C]lactate, [U-(14)C]2-phospho-L-lactate, or [8-(3)H]GTP were not successful owing to the instability of these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 min at 50 degrees C (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesis could be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F(420)-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F(420) follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B(12) and phospholipids.

Enzymes frequently rely on a broad repertoire of cofactors to perform chemically challenging transformations. The B6 coenzymes, composed of pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), are used by many transaminases, racemases, decarboxylases, and enzymes catalyzing alpha,beta and beta,gamma-eliminations. Despite the variety of reactions catalyzed by B6-dependent enzymes, the mechanism of almost all such enzymes is based on their ability to stabilize high-energy anionic intermediates in their reaction pathways by the pyridinium moiety of PLP/PMP. However, there are two notable exceptions to this model, which are discussed in this article. The first enzyme, lysine 2,3-aminomutase, is a PLP-dependent enzyme that catalyzes the interconversion of L-lysine to L-beta-lysine using a one-electron-based mechanism utilizing a [4Fe-4S] cluster and S-adenosylmethionine. The second enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase, is a PMP-dependent enzyme involved in the formation of 3,6-dideoxysugars in bacteria. This enzyme also contains an iron-sulfur cluster and uses a one-electron based mechanism to catalyze removal of a C-3 hydroxy group from a 4-hexulose. In both cases, the participation of free radicals in the reaction pathway has been established, placing these two B6-dependent enzymes in an exclusive class by themselves.

Glutamate mutase from Clostridium tetanomorphum binds coenzyme B(12) in a base-off/His-on form, in which the nitrogenous ligand of the B(12)-nucleotide function is displaced from cobalt by a conserved histidine. The effect of binding the B(12)-nucleotide moiety to MutS, the B(12)-binding subunit of glutamate mutase, was investigated using NMR spectroscopic methods. Binding of the B(12)-nucleotide to MutS was determined to occur with K(d)=5.6(+/-0.7) mM and to be accompanied by a specific conformational change in the protein. The nucleotide binding cleft of the apo-protein, which is formed by a dynamic segment with propensity for partial alpha-helical conformation (the "nascent" alpha-helix), becomes completely structured upon binding of the B(12)-nucleotide, with formation of helix alpha1. In contrast, the segment containing the conserved residues of the B(12)-binding Asp-x-His-x-x-Gly motif remains highly dynamic in the protein/B(12)-nucleotide complex. From relaxation studies, the time constant tau, which characterizes the time scale for the formation of helix alpha1, was estimated to be about 30 micros (15)N and was the same in both, apo-protein and nucleotide-bound protein. Thus, the binding of the B(12)-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fold. These results indicate MutS to be structured in such a way, as to be able to trap the nucleotide segment of the base-off form of coenzyme B(12) and provide, accordingly, the first structural clues as to how the process of B(12)-binding occurs.

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron–sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of −185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz= 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe−2S]+, [3Fe−4S]+, [4Fe−4S]+ or [4Fe−4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe−4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.

Adenosylcobalamin (AdoCbl) (coenzyme B 12 ) serves as a cofactor for enzymatic radical reactions. The recently solved X-ray structure of diol dehydratase in complex with cyanocobalamin (CN-Cbl) revealed the base-on mode of cobalamin binding. The active-site cavity inside the (β/α) 8 barrel seems to be a common molecular apparatus for coenzyme B 12 -dependent enzymes to spatially isolate highly reactive radical intermediates. Based on the direct ion–dipolar interactions between two hydroxyl groups of substrate and potassium ion in the active site and theoretical calculations, a new mechanism for diol dehydratase is proposed here in which a potassium ion participates directly in the catalysis. The mechanism of activation of the coenzyme's cobalt–carbon bond by which a catalytic radical is generated in the active site of diol dehydratase is also discussed on the basis of the conformation of the enzyme-bound cobalamin. It was highly suggested that the reactivity of the cobalt atom is controlled by the length of the CoN bond. Therefore, the role of the 5,6-dimethylbenzimidazole (DBI) moiety of cobalamin coenzyme in the enzyme catalysis is most likely to prevent the enzyme from mechanism-based inactivation by keeping the CoN bond distance long through steric repulsion between the flattened corrin ring and the base.

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B. The electron density at 1.6 A resolution of the M. barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations.

(Coβ-5′-Deoxyadenosin-5′-yl)-( p -cresyl)cobamide (Ado-PCC), a base-off analogue of coenzyme B 12 (Ado-Cbl), was used to elucidate the coenzyme B 12 binding mode of glutamate mutase, 2-methyleneglutarate mutase and ethanolamine ammonia–lyase (EAL). Ado-PCC functions as excellent coenzyme for the carbon skeleton rearrangements with apparent K m values of 200 and 10 nM for glutamate and 2-methyleneglutarate mutases, respectively. The corresponding values for Ado-Cbl are 60 and 54 nM, respectively. In contrast, Ado-PCC showed no coenzyme activity with EAL but was a competitive inhibitor with respect to Ado-Cbl. The K i value for Ado-PCC was 26 nM, the apparent K m value for Ado-Cbl was 30 nM. These results are in agreement with the notion that in glutamate and 2-methyleneglutarate mutases, a conserved histidine residue of the protein is coordinated to the cobalt atom of coenzyme B 12 , whereas in EAL the dimethylbenzimidazole residue of the coenzyme itself serves as ligand.

Two putativeMethanococcus jannaschiiisocitrate dehydrogenase genes, MJ1596 and MJ0720, were cloned and overexpressed inEscherichia coli, and their gene products were tested for the ability to catalyze the NAD- and NADP-dependent oxidative decarboxylation ofdl-threo-3-isopropylmalic acid,threo-isocitrate,erythro-isocitrate, and homologs ofthreo-isocitrate. Neither enzyme was found to use any of the isomers of isocitrate as a substrate. The protein product of the MJ1596 gene, designated AksF, catalyzed the NAD-dependent decarboxylation of intermediates in the biosynthesis of 7-mercaptoheptanoic acid, a moiety of methanoarchaeal coenzyme B (7-mercaptoheptanylthreonine phosphate). These intermediates included (−)-threo-isohomocitrate [(−)-threo-1-hydroxy-1,2,4-butanetricarboxylic acid], (−)-threo-iso(homo)2citrate [(−)-threo-1-hydroxy-1,2,5-pentanetricarboxylic acid], and (−)-threo-iso(homo)3citrate [(−)-threo-1-hydroxy-1,2,6-hexanetricarboxylic acid]. The protein product of MJ0720 was found to be α-isopropylmalate dehydrogenase (LeuB) and was found to catalyze the NAD-dependent decarboxylation of one isomer ofdl-threo-isopropylmalate to 2-ketoisocaproate; thus, it is involved in the biosynthesis of leucine. The AksF enzyme proved to be thermostable, losing only 10% of its enzymatic activity after heating at 100°C for 10 min, whereas the LeuB enzyme lost 50% of its enzymatic activity after heating at 80°C for 10 min.

Dimethylsulfoniopropionate (DMSP), an important compatible solute of many marine algae, can be metabolised by bacteria via cleavage to dimethylsulfide and acrylate or via an initial demethylation. This is the first report on the purification of an enzyme that specifically catalyses the demethylation of DMSP. The enzyme was isolated from the sulfate-reducing bacterium strain WN, which grows on DMSP and demethylates it to methylthiopropionate. DMSP:tetrahydrofolate (THF) methyltransferase from strain WN was purified 76-fold [to a specific activity of 40.5 μmol min −1 (mg protein) −1 ]. SDS polyacrylamide gel electrophoresis showed two bands of approximately 10 and 35 kDa; in particular the 35 kDa polypeptide became significantly enriched during the purification. Storage of the purified fraction at −20°C under nitrogen resulted in a 99% loss of activity in two days. The activity could be partially restored by addition of 200 μM cyanocobalamin, hydroxocobalamin or coenzyme B 12 . ATP did not have any positive effect on activity. Reduction of the assay mixture by titanium(III)nitrilotriacetic acid slightly stimulated the activity. Gel filtration chromatography revealed a native molecular mass between 45 and 60 kDa for the DMSP:THF methyltransferase. The enzyme was most active at 35°C and pH 7.8. Glycine betaine, which can be considered an N-containing structural analogue of DMSP, did not serve as a methyl donor for DMSP:THF methyltransferase. Various sulfur-containing DMSP-analogues were tested but only methylethylsulfoniopropionate served as methyl donor. None of these compounds inhibited methyl transfer from DMSP to THF. Strain WN did not grow on any of the sulfur-containing DMSP-analogues.

Background: Adenosylcobalamin (coenzyme B 12 ) serves as a cofactor for enzymatic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt–carbon bond of the coenzyme, although the mechanism of cleavage of its organometallic bond remains unsolved. Results: We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 A and 1.9 A resolution, respectively, at cryogenic temperatures. In the adeninylpentylcobalamin complex, the adenine ring is bound parallel to the corrin ring as in the free form and methylmalonyl-CoA-mutase-bound coenzyme, but with the other side facing pyrrole ring C. All of its nitrogen atoms except for N(9) are hydrogen-bonded to mainchain amide oxygen and amide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As compared with the cyanocobalamin complex, the sidechain of Serα224 rotates by 120° to hydrogen bond with N(3) of the adenine ring. Conclusions: The structure of the adenine-ring-binding site provides a molecular basis for the strict specificity of diol dehydratase for the coenzyme adenosyl group. The superimposition of the structure of the free coenzyme on that of enzyme-bound adeninylpentylcobalamin demonstrated that the tight enzyme–coenzyme interactions at both the cobalamin moiety and adenine ring of the adenosyl group would inevitably lead to cleavage of the cobalt–carbon bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5′-carbon radical accessible to the hydrogen atom of the substrate to be abstracted.

The universal force field (UFF) is used to minimize a series of cobalamins to (i) test the applicability of UFF to model unstrained cobalamin structures in comparison with the literature force field MM2′, a force field parameterized using a set of 19 cobalamin X-ray crystal structures, and (ii) to probe the steric-based, ground-state corrin butterfly effect, a literature hypothesis aimed at explaining the observed 10 12 enzymic acceleration of adenosylcobalamin (AdoCbl) CoC bond homolysis. The results obtained show that UFF is at least as good as MM2′ in modeling the five cobalamins which were originally used to test the accuracy of MM2′. Next, by using UFF to constrain the CoN(bzm) bond length in adenosylcobalamin (AdoCbl) to bond lengths ranging from 4.5 to 1.44 A, the putative axial-base-induced corrin butterfly conformation effect was modeled. The results show that while a short CoN(bzm) bond length does lengthen the CoC bond and increase the CoCC bond angle, the maximum magnitude of the predicted distortions appears, at least when considering steric effects alone, to be at most ∼1/3 that needed to accomplish the observed CoC bond homolysis acceleration. Equivalent results were obtained in analogous studies of base-free adenosylcobinamide (AdoCbi + ) in the presence of an exogenous N -methylimidazole base, [AdoCbi·N-MeIm] + . Overall, literature precedent, and now UFF molecular mechanics, all provide evidence against a solely axial-base-driven, sterically-induced corrin butterfly conformation effect as the dominant mechanism in the enzymic acceleration of AdoCbl CoC bond homolysis. Serious consideration must now be given to a combination of effects: an enzyme-induced rack mechanism (which can contain an enzyme -induced corrin butterfly conformational effect), and a coupling of Co⋯C cleavage, substrate or cysteine S⋯H bond cleavage, and Ado⋯H bond formation steps.

A series of [ω-(adenosin-5′-O- yl )alkyl]cobalamins were examined for their inhibitory properties of ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the presence of 5′-deoxyadenosylcobalamin (AdoCbl, Coenzyme B 12 ). These AdoCbl analogs, in which oligomethylene chains (C 3 -C 7 ) were inserted between the corrin Co-atom and a 5′-O-atom of the adenosine moiety, were designed to probe the Co-C bond posthomolysis state in AdoCbl-dependent enzymes, a state in which the Co and 5′-C distance is believed to be significantly increased. Experimentally, all five analogs were competitive inhibitors, with K i in the range of 8–56 μM. The [ω-(adenosin-5′-O- yl )alkyl]cobalamin analog with C 5 methylene carbons was the strongest inhibitor. This same pattern of inhibition, in which the C 5 -analog is the strongest inhibitor, was previously observed in the AdoCbl-dependent eliminase enzyme systems, diol dehydratase and glycerol dehydratase. However, in methylmalonyl CoA mutase , the strongest inhibition is by the C 6 -analog. This supports the hypothesis that the cobalamin posthomolysis intermediate in the eliminase enzymes differs from that in the mutase enzymes. These findings led, in turn, to an examination of the visible spectra of enzyme-bound cob(II)alamin in these two subclasses of AdoCbl-dependent enzymes. The results reveal an additional insight into the difference between the two classes: in the eliminases , the γ-band of bound cob(II)alamin is shifted from the 473 nm for free cob(II)alamin to longer wavelengths, 475–480 nm. However, in mutases , the γ-band of bound cob(II)alamin is shifted to shorter wavelengths, 465–470 nm. Overall, the results (a) provide strong evidence that two subclasses of AdoCbl-dependent enzymes exist, (b) give insights into the probable posthomolysis state in RTPR and other eliminases, and (c) identifies the C 5 -analog as the tightest-binding analog for crystallization and other biophysical studies.

The coenzyme B 12 -dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n -butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n - and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis . Mutants prepared by inserting a hygromycin resistance gene ( hygB ) into either icmA or mutB , encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA :: hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB :: hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. 13 C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n -butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.

The syntheses, isolation and characterization of cyclohexylthiolatocobalamin (C6H11SCbl), glutathionylcobalamin (GluSCbl), and cysteinylcobalamin (CysSCbl) are reported in 75, 55, and 65% yield, respectively. Characterization was achieved using elemental analyses, L-SIMS (liquid secondary ion mass spectrometry), UV-visible spectroscopy and, for the more stable C6H11SCbl and GluSCbl, our recently established 1H NMR method (which emphasizes the readily interpreted aromatic region of the cobalamin's 1H NMR spectrum). Preliminary evidence is presented for clean homolysis of the RS-Co bond in C6H11SCbl, GluSCbl, and CysSCbl to give RS. and .Co(II)Cbl radical pairs analogous to those that are intermediates in ribonucleoside triphosphate reductase (RTPR). A summary is provided which emphasizes the seven variables identified to date, underlying the successful syntheses and isolation of thiolatocobalamins, variables which make the one-step syntheses of RSCbls considerably more complex than they initially appear. Also briefly discussed are the analogous protein-S-Cbl complexes that are seen as side-products in RTPR, and the probability that such side-products are formed when HOCbl.HX is used as a possible 'active-site inhibitor' complex with B12-dependent enzymes.

Background: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B 12 ) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt–carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an α 2 β 2 γ 2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase–cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. Results: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers ( α β γ ) 2 . The cobalamin molecule is bound between the α and β subunits in the ‘base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The α subunit includes a ( β / α ) 8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. Conclusions: This is the first crystallographic indication of the ‘base-on' mode of cobalamin binding. An unusually long cobalt–base bond seems to favor homolytic cleavage of the cobalt–carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

Anaerobic respiration with elemental sulfur/polysulfide or organic disulfides is performed by several bacteria and archaea, but has only been investigated in a few organisms in detail. The electron transport chain that catalyzes polysulfide reduction in the Gram-negative bacterium Wolinella succinogenes consists of a dehydrogenase (formate dehydrogenase or hydrogenase) and polysulfide reductase. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the periplasm. The mechanism of electron transfer from formate dehydrogenase or hydrogenase to polysulfide reductase is discussed. The catalytic subunit of polysulfide reductase belongs to the family of molybdopterin-dinucleotide-containing oxidoreductases. From the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 an integral membrane complex has been isolated which catalyzes the reduction of sulfur with H2 as electron donor. This enzyme complex, which is composed of a hydrogenase and a sulfur reductase, contains heme groups and several iron-sulfur clusters, but does not contain molybdenum or tungsten. In methanogenic archaea, the heterodisulfide of coenzyme M and coenzyme B is the terminal electron acceptor of the respiratory chain. In methanogens belonging to the order Methanosarcinales, this respiratory chain is composed of a dehydrogenase, the membrane-soluble electron carrier methanophenazine, and heterodisulfide reductase. The catalytic subunit of heterodisulfide reductase contains only iron-sulfur clusters. An iron-sulfur cluster may directly be involved in the reduction of the disulfide substrate.

Radicals are reactive intermediates of growing importance in enzymatic catalysis. There are reactions in which neutral radicals participate and those where radical anions are involved. The former class is illustrated by lysine 2,3-aminomutase and also by enzymes dependent on coenzyme B12, that catalyse carbon skeleton rearrangements (e.g. glutamate mutase). A substrate-based radical for both lysine 2,3-aminomutase and glutamate mutase has been characterised by EPR spectroscopy. Representatives of the second class are 2-hydroxyglutaryl-CoA dehydratase, benzoyl-CoA reductase, DNA photolyase and chorismate synthase, all of which may generate the radical anion by one-electron reduction. 4-Hydroxybutyryl-CoA dehydratase, pyruvate formate lyase, and the coenzyme B12-dependent eliminases (ribonucleotide reductase, ethanolamine ammonia lyase and diol dehydratase) could be examples of radical anion formation by one-electron oxidation. The electron-rich ketyl-like radical anions cause the elimination of an adjacent group. The advantages of using radicals as intermediates in enzymatic transformations are their high reactivity and special properties. However, this reactivity includes rapid bimolecular combination with dioxygen and radicals are therefore primarily utilised as intermediates by anaerobic organisms.

One subclass of B 12 -requiring enzymes is now known to bind their B 12 coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B 12 analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi + s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH 2 Cbi + and CF 3 Cbi + , in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.

The genes encoding coenzyme B 12 -dependent glycerol dehydratase of Clostridium pasteurianum were subcloned and expressed in Escherichia coli . The native molecular mass of the enzyme is 190 000 Da. The enzyme converts glycerol, 1,2-propanediol and 1,2-ethanediol to 3-hydroxypropionaldehyde, propionaldehyde and acetaldehyde, respectively, but glycerol is the preferred substrate. The nucleotide sequences of the dhaBCE genes encoding the three subunits of glycerol dehydratase and of orfZ whose function is unknown were determined. The deduced products of the dhaBCE genes with calculated molecular masses of 60 813, 19 549 and 16 722 Da, respectively, revealed high similarity to amino acid sequences of subunits of coenzyme B 12 -dependent glycerol and diol dehydratases from other organisms.

5′-Deoxyadenosyl-13-epicobalamin(Ado-13-epiCbl) is a coenzymatically active structural analog of coenzyme B 12 (5′-deoxyadenosylcobalamin, AdoCbl) in which epimerization at the corrin ring C13 places the e propionamide side chain in an “upwardly” axial position. This would seem to bring the e side chain into direct steric contact with the adenosyl (Ado) moiety in its normal “southern” conformation (with the purine above the C ring) seen in the solid state for AdoCbl. Molecular mechanics calculations show that this conformation is the global minimum for AdoCbl, but that three other conformations represent local minima, including a “western”, a “northern”,and an “eastern” conformation, the last of which is known to be populated at room temperature from NMR studies. A structure for Ado-13-epiCbl has been generated by molecular dynamics/simulated annealing calculations starting from the neutron diffraction coordinates for AdoCbl after epimerization at C13. Molecular mechanics calculations on the resulting structure show that a “western” conformation is the global minimum, but that three other conformations, a “northern”, an “eastern”, and a “northeastern”, are of relatively low energy and separated by relatively low energy barriers. The traditional “southern” conformation does not appear as a local energy minimum and is some 6 kcal mol −1 destabilized relative to the global minimum due to geometric distortions to relieve steric strain. Two-dimensional homonuclear NMR experiments show extraordinary conformational flexibility in Ado-13-epiCbl with 21 NOE crosspeaks between Ado protons and non-Ado protons, 50% more than in AdoCbl itself. Together with the calculated geometries for the four Ado-13-epiCbl conformers, these data suggest that at least three, and possibly all four of the conformers are populated at room temperature, in contrast to AdoCbl, in which only the “southern” and “eastern” conformers are populated under these conditions. Considering the enzymology of Ado-13-epiCbl, the inaccessibility of its “southern” conformation, and the activity of other AdoCbl analogs structurally modified in the Ado moiety, it is suggested that it may well be the “eastern” conformation, accessible to both AdoCbl and Ado-13-epiCbl, which is enzymatically active.