Cleavage Of Co-C Bond Research Articles

Biologically inspired Co-MOF as catalase mimics with an unexpected ROS production ability for the efficient removal of organic dyes

Developing an efficient advanced oxidation technology is of great significance for water treatment and environmental protection. Here, inspired by the structure and feature of coenzyme B12 in natural enzymes (cleavage of Co-C bond triggers the generation of radical), a Co based metal-organic framework (Co-MOF) with catalase-mimicking activity was synthesized and applied in the degradation of organic dyes. Firstly, Co-MOF nanozyme was fabricated via the coordination self-assembly strategy at room temperature. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive scanning (EDS), powder X-ray diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the morphology and structure. The results showed that the obtained Co-MOF nanozyme was rhombic dodecahedral morphology with a size of ∼ 500 nm and excellent crystallinity. The enzymatic experiments proved that such nanozyme possessed a single catalase-like activity. Further research results demonstrated that Co-MOF-based degradation process of acid fuchsin (AF) or crystal violet (CV) had excellent pH adaptability (pH > 5.0). Unexpectedly, an ultra-high catalytic degradation rate (211.35 min−1 g−1 L) was successfully achieved in the catalytic degradation of AF, and it was approximately 5∼200 times of the previous optimal nanozyme or catalysts. In complex matrices and real wastewater, this catalytic process could still maintain degradation efficiency above 90% or even higher within 5 minutes, proving its excellent stability and adaptability. The results of radical and electron quenching experiments, electron paramagnetic resonance (EPR), and electrochemical analysis demonstrated that the excellent catalytic degradation performance was attributed to the generation of reactive oxygen species (ROS) (∙O2 was the main contributor) and the existence of electron transfer. In summary, this new catalytic property endows Co-MOF nanozyme with considerable potential for dye degradation, extending the application scope of catalase mimics.

Redox driven B12-ligand switch drives CarH photoresponse

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.

Synthesis and structural characterization of new Schiff base probe: A conformation modulator for Co-C bond cleavage in coenzyme B12-dependent enzymes

Coenzyme B12-dependent enzymes catalyze complex radical-mediated reactions initiated by homolytic cleavage of the organometallic Co-C bond of the cofactors upon substrate binding. Herein, we synthesize new 2,3-diaminomaleonitrile (DAMN) based Schiff base probe for an investigation of binding interaction with coenzyme B12-dependent enzymes by molecular docking approach. The formulated compound has been well characterized by elemental analysis, NMR (1H and 13C), Mass spectrometry and UV-visible spectroscopy. Moreover, the complete structure elucidation of Schiff base probe was achieved via X-ray crystallography. Docking study reveals that the newly synthesized Schiff base compound confers good binding response towards glutamate mutase and methionine synthase as supported by calculated docking score and binding energies -7.7 kcal/mol and -8.9 kcal/mol, respectively. The results indicate significant binding effect of synthesized Schiff base on the catalysis of B12-dependent enzymes. We hope that the present information can be utilized to develop potent therapeutic drugs.

Cobalt-Mediated Switchable Catalysis for the One-Pot Synthesis of Cyclic Polymers.

A cobalt salen pentenoate complex [salen=(R,R)-N,N'-bis(3,5-di-tertbutylsalicylidene)-1,2-cyclohexanediamine] is rationally designed as the catalyst for the ring-opening copolymerization (ROCOP) of epoxides/anhydrides/CO2 . Via migratory insertion of carbon monoxide (CO) into the Co-O bonds, the ROCOP-active species α-alkene-ω-O-CoIII (salen) can be rapidly and quantitatively transformed into α-alkene-ω-O2 C-CoIII (salen) telechelic linear precursors. Upon dilution of reaction mixtures, the homolytic cleavage of Co-C bonds induced by visible light generates α-alkene acyl radicals that spontaneously undergo intramolecular radical addition to afford organocobalt-functionalized cyclic polyesters and CO2 -based polycarbonates with excellent regioselectivity. The cyclic products can either react with radical scavengers to generate metal-free cyclic polymers or serve as photo-initiators for organometallic-mediated radical polymerization (OMRP) to produce tadpole-shaped copolymers.

The Electronic Structure and Mechanism of the Adenosylcobalamin-Dependent Bio-Processes as Determined by the MCSCF Method

The CASSCF geometry optimization of the adenosylcobalamin cofactor dependent processes common models with 12 orbitals and 12 electrons in the active space has been performed. The MCSCF geometry optimization shows a strong HOMO-LUMO coupling during the CASSCF orbitals mixing process. The HOMO-LUMO coupling causes an electronic density transfer from the HOMO, which at the beginning of the optimization process is constituted from the substrate atoms orbitals, to the LUMO, which is constituted from the adenosylco(III)balamin structure atomic orbitals. The Co-C bond cleavage reaction is starting from the beginning of the geometry optimization process due to the intermolecular transferred electronic density from the substrates to the adenosylco(III)balamin cofactor compound. Then, the HOMO and LUMO of the calculated models are converting into a bonding and an antibonding pair of orbitals with a central atom plus σ-axial ligands orbitals contribution and with a corrin ring plus axial ligands orbitals contribution, respectively. The HOMO-LUMO mixing process in the CASSCF procedure causes the intermolecular charge transfer process that converts into intramolecular charge transfer process, which is increasing up to about 1e- at the Co-C bond cleavage distance. The substrates of the adenosylcobalamin cofactor dependent bio-processes from one side and the 5'- deoxy-5’-adenosyl radical from another side are permanently growing their direct interactions along with the Co-C bond rupture process up to a strong direct interaction at the Co-C bond cleavage distance. Evidently, this is allowing for a hydrogen atom transfer between them. Altogether, the total energy barrier of the hydrogen transfer reaction from the substrate to the 5'- deoxy-5’-adenosyl radical reaction, the CASSCF HOMO and LUMO surface orbitals of the substrate and 5'-adenosyl radical interaction common model before and after the hydrogen transfer and a strong Pseudo-Jahn-Teller effect for only direct reaction demonstrate that the hydrogen transfer is an irreversible tunneling process, which certainly leads to the final products. All these results are pointing out to the Co-C bond cleavage and hydrogen transfer from substrate to 5'- deoxy-5’-adenosyl ligand concerted reactions in full agreement with the experimental data.

Highly active Mo-V-based bifunctional catalysts for catalytic conversion of lignin dimer model compounds at room temperature

A novel strategy was proposed for the cleavage of COC bonds in β-O-4 lignin substrate. 2-(2′-methoxyphenoxyl)-1-phenylethanol was used as a lignin dimer model compound with a β-O-4 ether linkage and a hydroxyl group on α-C position. Mo-V-based bifunctional catalysts have been developed to selectively catalyze the oxidation of Cα-OH and depolymerize β-O-4 dimers under mild reaction conditions. In addition, the catalysts were characterized by XRD, SEM, TPR, NH3-TPD and N2 adsorption. At room temperature, the conversion of dimer reached up to 89% within 4 h. Based on the analysis of HPLC-MS results, a two-step oxidation-cleavage pathway for lignin depolymerization was proposed. In the catalytic reaction process, the Cα-OH in the lignin dimer was first oxidized to obtain Cα ketone, and then the Cα ketone intermediate was further catalyzed to break the β-O-4 bond. In the study, aromatic monomer compounds were obtained under the Mo-V-based bifunctional catalysts.

Photolytic Cleavage of Co-C Bond in Coenzyme B12-Dependent Glutamate Mutase.

Glutamate mutase (GLM) is a coenzyme B12-dependent enzyme that catalyzes the conversion of S-glutamate to (2 S,3 S)-3-methyl aspartate. The initial step in the catalytic process is the homolytic cleavage of the coenzyme's Co-C bond upon binding of a substrate. Alternatively, the Co-C bond can be cleaved using light. To investigate the photolytic cleavage of the Co-C bond in GLM, we applied a combined density functional theory/molecular mechanics (DFT/MM) and time-dependent-DFT/MM method to scrutinize the ground and the low-lying excited states. Potential energy surfaces (PESs) were generated as a function of axial bond lengths to describe the photodissociation mechanism. The S1 PES was characterized as the crossing of two electronic states, metal-to-ligand charge transfer (MLCT), and ligand field (LF). In GLM, radical pairs generate from the LF state. Two pathways, path A and path B, were identified as possible channels to connect the MLCT and LF electronic states. The S1 PES in GLM was compared with the S1 PES for coenzyme B12-dependent ethanolamine ammonia lyase as well as the isolated AdoCbl cofactor. Finally, the theoretical insights related to the photodissociation mechanism were compared with transient absorption spectroscopy, electron paramagnetic resonance, and resonance Raman spectroscopy.

Ruthenium trichloride catalyzed conversion of cellulose into 5-hydroxymethylfurfural in biphasic system

The production of 5-hydroxymethylfurfural (5-HMF) from cellulose catalyzed by a series of transition metal chlorides (i.e. FeCl3, RuCl3, VCl3, TiCl3, MoCl3 and CrCl3) was studied in biphasic system. RuCl3 was the most efficient catalyst among these transition metal chlorides for 5-HMF production, and resulted in both the highest yield of 83.3% and selectivity of 87.5% in NaCl-aqueous/butanol biphasic system. XRD analysis and FTIR spectroscopy were applied to further characterize the RuCl3 catalyzed cellulose slurries to reveal the catalytic reaction mechanism. Results demonstrated that RuCl3 enhanced the decrystallization and cleavage of COC bonds in cellulose, promoted the subsequent dehydration of glucose into 5-HMF, while suppressed the glucose retro-aldol reaction to byproduct lactic acid. In addition, with the assistance of NaCl-aqueous/butanol biphasic system, 5-HMF further degradation was limited and thusly maintained a desired 5-HMF yield. This proposed approach provides an efficient strategy for one-pot conversion of cellulose into 5-HMF.

The Nature of the Co-C Bond Cleavage Processes in Methylcob(II)Alamin and Adenosylcob(III)Alamin

The Nature of the Co-C Bond Cleavage Processes in Methylcob(II)Alamin and Adenosylcob(III)Alamin

The molecular mechanism of the open-closed protein conformational cycle transitions and coupled substrate binding, activation and product release events in lysine 5,6-aminomutase.

How a protein domain motion is coupled to the catalytic cycle is a current subject in enzymology. We render down a complicated domain motion in the 5'-deoxyadenosylcobalamin and pyridoxal-5'-phosphate codependent radical enzyme, lysine 5,6-aminomutase, into dominant contributions from Lys370α and Asp298α to the critical Co-C bond cleavage trigger and open-closed cycle transitions.

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Spectroscopic and computational studies of cobalamin species with variable lower axial ligation: implications for the mechanism of Co-C bond activation by class I cobalamin-dependent isomerases.

5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor's Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor's electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.

Synthesis and properties of formylcobalamin and propionylcobalamin, novel acylcobalamins.

Formylcobalamin (formyl-Cbl), a C1-unit carrying corrinoid, and propionylcobalamin (propionyl-Cbl) were synthesized for the first time, and their properties were compared with those of acetylcobalamin (acetyl-Cbl). Formyl-Cbl, acetyl-Cbl, and propionyl-Cbl were decomposed by a NH2OH treatment, forming formo-, aceto-, and propionohydroxamic acids, respectively, which offers a proof for the presence of "activated" acyl groups and for their structures of Co-acyl-Cbls. These results, together with chromatographic, electrophoretic, and spectroscopic properties, indicate that the acyl-Cbls synthesized are actually formyl-Cbl, acetyl-Cbl, and propionyl-Cbl. Spectroscopic and electrophoretic properties were consistent with the σ-donor strength or trans-effect increasing in the order: formyl<acetyl<propionyl. All acyl-Cbls underwent the Co-C bond cleavage upon photo-irradiation with a tungsten light bulb as well as upon treatment with alkali, forming aquacobalamin (aqCbl) and hydroxocobalamin (OH-Cbl), respectively, under air. Formyl-Cbl was thermally unstable and decomposed to aqCbl, and the thermal stability in neutral solution was much lower than in diluted HCl. In contrast, acetyl-Cbl and propionyl-Cbl were heat-stable under these conditions. Formyl-Cbl was essentially unsusceptible to unbufferized KCN solution, although acetyl-Cbl and propionyl-Cbl were rapidly converted to dicyanocobalamin under the same conditions. Such abnormal behaviors of formyl-Cbl suggest that it mainly exists in a hydrated form as formaldehyde.

Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2 with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO(-) group at residue α287 was absolutely required for activity and coenzyme Co-C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co-C bond homolysis, stabilizes the transition state for the migration of NH2 from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2 migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+). The activity was very sensitive to the position of -COO(-) at α362. Aspα362 would assist Co-C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+) and electrostatic interaction.

TD-DFT insight into photodissociation of the Co-C bond in coenzyme B12.

Coenzyme B12 (AdoCbl) is one of the most biologically active forms of vitamin B12, and continues to be a topic of active research interest. The mechanism of Co-C bond cleavage in AdoCbl, and the corresponding enzymatic reactions are however, not well understood at the molecular level. In this work, time-dependent density functional theory (TD-DFT) has been applied to investigate the photodissociation of coenzyme B12. To reduce computational cost, while retaining the major spectroscopic features of AdoCbl, a truncated model based on ribosylcobalamin (RibCbl) was used to simulate Co-C photodissociation. Equilibrium geometries of RibCbl were obtained by optimization at the DFT/BP86/TZVP level of theory, and low-lying excited states were calculated by TD-DFT using the same functional and basis set. The calculated singlet states, and absorption spectra were simulated in both the gas phase, and water, using the polarizable continuum model (PCM). Both spectra were in reasonable agreement with experimental data, and potential energy curves based on vertical excitations were plotted to explore the nature of Co-C bond dissociation. It was found that a repulsive 3(σCo−C → σ*Co−C) triplet state became dissociative at large Co-C bond distance, similar to a previous observation for methylcobalamin (MeCbl). Furthermore, potential energy surfaces (PESs) obtained as a function of both Co-CRib and Co-NIm distances, identify the S1 state as a key intermediate generated during photoexcitation of RibCbl, attributed to a mixture of a metal-to-ligand charge transfer (MLCT) and a σ bonding-ligand charge transfer (SBLCT) states.

Redesign of coenzyme B12 dependent diol dehydratase to be resistant to the mechanism‐based inactivation by glycerol and act on longer chain 1,2‐diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract • Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).

Initial step of B12-dependent enzymatic catalysis: energetic implications regarding involvement of the one-electron-reduced form of adenosylcobalamin cofactor

Density functional theory analysis was performed to elucidate the impact of one-electron reduction upon the initial step of adenosylcobalamin-dependent enzymatic catalysis. The transition state (TS) corresponding to the Co-C bond cleavage and subsequent hydrogen abstraction from the substrate was located. The intrinsic reaction coordinate calculations predicted that the reaction consisting of Co-C5' bond cleavage in [Co(III)(corrin(•))]-Rib (where Rib is ribosyl) and hydrogen-atom abstraction from the CH(3)-CH(2)-CHO substrate occurs in a concerted fashion. The computed activation energy barrier of the reaction (15.0kcal/mol) was lowered by approximately 54.5% in comparison with the reaction involving the positively charged cofactor model (Im-[Co(III)(corrin)]-Rib(+), where Im is imidazole; energy barrier =33.0kcal/mol). The Im base was detached during the TS search in the reaction involving the one-electron-reduced analogue. Thus, to compare the energetics of the two reactions, the axial Im ligand detachment energy for the Im-[Co(III)(corrin(•))]-Rib model was computed [7.6kcal/mol (gas phase); 4.6kcal/mol (water)]. Consequently, the effective activation energy barrier for the reaction mediated by the Im-off [Co(III)(corrin(•))]-Rib was estimated to be 22.6kcal/mol, which implied an overall 31.5% reduction in the energetic demands of the reaction. Considering that the lengthened Co-N(axial) bond has been observed in X-ray crystal structure studies of B(12)-dependent mutases, the catalytic impact induced by one-electron reduction of the cofactor is expected to be higher in the presence of the enzymatic environment.

Diol dehydratase‐reactivating factor is a reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase

Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg(2+) by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg(2+). Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg(2+). Upon complex formation, one β subunit was released from the (αβ)₂ tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD β subunit and the reactivase β subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

Structural Basis for Adenosylcobalamin Activation in AdoCbl-Dependent Ribonucleotide Reductases

Class II ribonucleotide reductases (RNR) catalyze the formation of an essential thiyl radical by homolytic cleavage of the Co-C bond in their adenosylcobalamin (AdoCbl) cofactor. Several mechanisms for the dramatic acceleration of Co-C bond cleavage in AdoCbl-dependent enzymes have been advanced, but no consensus yet exists. We present the structure of the class II RNR from Thermotoga maritima in three complexes: (i) with allosteric effector dTTP, substrate GDP, and AdoCbl; (ii) with dTTP and AdoCbl; (iii) with dTTP, GDP, and adenosine. Comparison of these structures gives the deepest structural insights so far into the mechanism of radical generation and transfer for AdoCbl-dependent RNR. AdoCbl binds to the active site pocket, shielding the substrate, transient 5'-deoxyadenosyl radical and nascent thiyl radical from solution. The e-propionamide side chain of AdoCbl forms hydrogen bonds directly to the α-phosphate group of the substrate. This interaction appears to cause a "locking-in" of the cofactor, and it is the first observation of a direct cofactor-substrate interaction in an AdoCbl-dependent enzyme. The structures support an ordered sequential reaction mechanism with release or relaxation of AdoCbl on each catalytic cycle. A conformational change of the AdoCbl adenosyl ribose is required to allow hydrogen transfer to the catalytic thiol group. Previously proposed mechanisms for radical transfer in B12-dependent enzymes cannot fully explain the transfer in class II RNR, suggesting that it may form a separate class that differs from the well-characterized eliminases and mutases.