Cobalamin Research Articles

Examination of the Glycine Betaine-Dependent Methylotrophic Methanogenesis Pathway: Insights Into Anaerobic Quaternary Amine Methylotrophy

Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon Methanolobus vulcani B1d metabolizes glycine betaine (GB) through a corrinoid-dependent GB:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of M. vulcani B1d revealed a gene encoding a predicted non-pyrrolysine MttB homolog (MV8460) with high sequence similarity to the GB methyltransferase encoded by Desulfitobacterium hafniense Y51. MV8460 catalyzes GB-dependent methylation of free cob(I)alamin indicating it is an authentic MtgB enzyme. Proteomic analysis revealed that MV8460 and a corrinoid binding protein (MV8465) were highly abundant when M. vulcani B1d was grown on GB relative to growth on trimethylamine. The abundance of a corrinoid reductive activation enzyme (MV10335) and a methylcorrinoid:CoM methyltransferase (MV10360) were significantly higher in GB-grown B1d lysates compared to other homologs. The GB:CoM pathway was fully reconstituted in vitro using recombinant MV8460, MV8465, MV10335, and MV10360. Demonstration of the complete GB:CoM pathway expands the knowledge of direct QA-dependent methylotrophy and establishes a model to identify additional ecologically relevant anaerobic quaternary amine pathways.

Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase.

The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of L-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5'-deoxyadenosyl 5'-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.

Elucidating the mechanism of cob(I)alamin mediated methylation reactions by alkyl halides: SN2 or radical mechanism?

Methyl transfer reactions mediated by cobalamins (Cbls) have been considered as one of the most important biologically relevant molecular transformations for many enzymatic reactions catalyzed by Cbl-dependent enzymes. The exact mechanism of methyl transfer reactions involving Cbls is still poorly understood. To investigate the mechanistic details of Cbl mediated methylations by alkyl halides, density functional theory (DFT) along with the polarizable continuum model (PCM/water) for solvation has been applied. Two different mechanisms have been examined, namely SN2 and radical-based electron transfer (ET) to elucidate the methyl transfer reaction. The calculations have suggested that the methyl transfer from methyl halides proceeds through SN2 nucleophilic displacement. However, with more bulky alkyl substrate, namely isopropyl and tert-butyl halides the reaction followed the ET-based radical pathway which is associated with an ET from diradical form of cob(I)alamin to alkyl halides. Our proposed mechanism for alkyl transfer reaction corroborates with the experimental findings, which reported a mechanistic switch from a two-electron (SN2-type) to a one-electron mechanism for sterically demanding alkyl halides. The present theoretical contribution provides more in-depth insight into the methyl transfer reaction catalyzed by corrinoid-dependent methyltransferases.

Probing the Excited State of Methylcobalamin Using Polarized Time-Resolved X-ray Absorption Spectroscopy.

We use picosecond time-resolved polarized X-ray absorption near-edge structure (XANES) measurements to probe the structure of the long-lived photoexcited state of methylcobalamin (MeCbl) and the cob(II)alamin photoproduct formed following photoexcitation of adenosylcobalamin (AdoCbl, coenzyme B12). For MeCbl, we used 520 nm excitation and a time delay of 100 ps to avoid the formation of cob(II)alamin. We find only small spectral changes in the equatorial and axial directions, which we interpret as arising from small (<∼0.05 Å) changes in both the equatorial and axial distances. This confirms expectations based on prior UV-visible transient absorption measurements and theoretical simulations. We do not find evidence for the significant elongation of the Co-C bond reported by Subramanian [ J. Phys. Chem. Lett. 2018 , 9 , 1542 - 1546 ] following 400 nm excitation. For AdoCbl, we resolve the difference XANES contributions along three unique molecular axes by exciting with both 540 and 365 nm light, demonstrating that the spectral changes are predominantly polarized along the axial direction, consistent with the loss of axial ligation. These data suggest that the microsecond "recombination product" identified by Subramanian et al. is actually the cob(II)alamin photoproduct that is produced following bond homolysis of MeCbl with 400 nm excitation. Our results highlight the pronounced advantage of using polarization-selective transient X-ray absorption for isolating structural dynamics in systems undergoing atomic displacements that are strongly correlated to the exciting optical polarization.

Seminal but not Serum Levels of Holotranscobalamin are Altered in Morbid Obesity and Correlate with Semen Quality: A Pilot Single Centre Study.

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility (r = 0.394, p = 0.012), sperm concentration (r = 0.401, p = 0.009), total sperm number (r = 0.343, p = 0.028), and negative correlation with semen pH (r = −0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

Biochemical Characterization of the Methylmercaptopropionate:Cob(I)alamin Methyltransferase from Methanosarcina acetivorans.

Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene ( cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by ΛC/Cl, the slope of δ13C versus δ37Cl regressions) was recently recognized to reveal different reduction mechanisms with vitamin B12 as a model reactant for reductive dehalogenase activity. Large ΛC/Cl values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller ΛC/Cl values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large ΛC/Cl values for cis-DCE (ΛC/Cl = 10.0 to 17.8) that contrasted with identical smaller ΛC/Cl for TCE and PCE (ΛC/Cl = 2.3 to 3.8). For TCE, the trend of small ΛC/Cl could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (ΛC/Cl = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of ΛC/Cl are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE.

Mechanistic studies on the reaction between glutathionylcobalamin and selenocysteine

The reaction between glutathionylcobalamin (GSCbl), a complex of Co(III)-cobalamin with glutathione, and selenocysteine (Sec) was investigated using ultraviolet-visible (UV–vis) spectroscopy. The interaction results in the formation of cob(II)alamin and proceeds via two pathways: (i) a rapid formation of complex between GSCbl and Sec followed by the rate-determining substitution of glutathionyl-ligand by Sec and rapid electron-transfer from Se-atom to Co(III)-ion and (ii) a nucleophilic attack of Co(III)-S bond by Sec.

Mechanistic Implications of Reductive Co-C Bond Cleavage in B12-Dependent Methylmalonyl CoA Mutase.

Vitamin B12-dependent enzymes catalyze several difficult radical reactions. There are fundamental open questions that need to be addressed to fully understand the formation of highly reactive radical species, its dynamics, and interaction with the substrate and enzyme. In this work, ab initio molecular dynamics was performed within a QM/MM framework on a reduced AdoCbl cofactor, which was taken as a post proton-coupled electron transfer initial step for the activation of the AdoCbl-dependent methylmalonyl CoA mutase enzyme. The calculated free-energy profile reveals two possible pathways, stepwise (I) and concerted (II) for the reductive Co-C cleavage and subsequent H-abstraction. The computed activation barrier from metadynamics for both the pathways is comparable (78.5 and 76.2 kJ/mol, respectively); however, the concerted pathway may be preferred kinetically because it avoids the formation of a high-energy radical intermediate with possibly a larger recrossing rate. Our results are consistent with the previous conductor hypothesis, indicating the explicit role of cob(II)alamin in stabilizing the radical intermediate involved in the H-atom transfer.

Light-Induced Activation of Organo-Metallic Co-C Bond in MeCbl-Dependent Methionine Synthase- QM/MM Study

Methylcobalamin (MeCbl)-dependent enzyme methionine synthase (MetH), plays a critical role in the catalysis of methyl group transfer from methyltetrahydrofolate to homocysteine. It often performs a side reaction to generate cob (II)alamin through photolysis of organometallic Co-C bond. A hybrid QM/MM method (DFT/MM and TD-DFT/MM) has been performed to explore the photochemistry of MeCbl-bound MetH. The manifold of low-lying excited states and corresponding potential energy surface (PES) of electronically excited S1 state has been constructed as a function of axial bond lengths to elucidate the mechanism of photoinduced activation of Co-C bond inside enzyme to investigate the photolytic properties of MeCbl-bound MetH. The analysis of S1 PES has revealed that the two different electronic states, namely metal-to-ligand charge transfer (MLCT) and the ligand field (LF), of S1 PES are relevant to the photodissociation of Co-C bond. There are two possible pathways identified, Path A and Path B, that connect the MLCT to LF state that represent possible photodissociation mechanisms. In the case of MetH, one possible photodissociation pathway (Path B) was identified based on the energetics of MLCT and LF states.The energetically accessible Path B involves initial the detachment of Co-NIm followed by a displacement of the Co-C bond prior the formation of cob(II)alamin / CH3 radical pair (RP). The photochemical data of base-on MeCbl was compared with the computed result of MeCbl-bound MetH to understand the effect of enzymatic environment on the photolytic properties of MeCbl. In compare to MeCbl in solution, the LF state is energetically higher in energy inside the enzyme which makes it easier to form RP for isolated cofactor in solution. Therefore the enzymatic environment has played a role in the destabilization of the LF state which impede the photolysis of MeCbl-bound MetH.

Mechanism of the photo-induced activation of Co[sbnd]C bond in methylcobalamin-dependent methionine synthase

Methylcobalamin (MeCbl)-dependent enzyme methionine synthase (MetH), plays a critical role in the catalysis of methyl group transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. It often performs a side reaction to generate cob(II)alamin through photolysis of the organometallic CoC σ bond. A hybrid QM/MM method has been applied to explore the photochemistry of MeCbl-bound MetH. The photolytic properties of MeCbl inside MetH are mediated by its manifold of low-lying excited states. The corresponding potential energy surfaces (PESs) of the electronically excited S1 state has been constructed as a function of axial bond lengths to elucidate the mechanism of photo-induced activation of CoC bond inside the enzyme. The analysis of the S1 PES has revealed that the two different electronic states of the S1 PES, namely metal-to-ligand charge transfer (MLCT) and the ligand field (LF), are relevant to the photodissociation of the CoC bond. There are two possible pathways identified, Path A and Path B, that connect the MLCT to LF state that represent possible photodissociation mechanisms. In the case of MetH, one possible photodissociation pathway (Path B) was identified based on the energetics of the MLCT and LF states. The energetically accessible Path B involves the initial detachment of the Co-NIm bond followed by a subsequent displacement of the CoC bond prior to the formation of cob(II)alamin / CH3 radical pair (RP). The photochemical data of base-on MeCbl in solution was compared with the computed result of MeCbl-bound MetH to understand the effect of the enzymatic environment on the photolytic properties of MeCbl.

Off to the Races: Comparison of Excited State Dynamics in Vitamin B12 Derivatives Hydroxocobalamin and Aquocobalamin.

Ultrafast time-resolved spectroscopy was used to study the photochemistry of hydroxocobalamin (HOCbl) and aquocobalamin (H2OCbl+) in solution. Spectroscopic measurements and TD-DFT simulations provide a consistent picture of the spectroscopy and photochemistry. Excitation of H2OCbl+ results in formation of an excited state followed by rapid internal conversion to the ground state (0.35 ± 0.15 ps) through an S1/S0 seam at a slightly elongated Co-O bond length and a significantly elongated Co-NIm bond length. In contrast, the initial elongation of the axial bonds in HOCbl is followed by contraction to an excited state minimum with bonds slightly shorter than those in the ground state. Internal conversion to the ground state follows on a picosecond time scale (5.3 ± 0.4 ps). For both compounds, photodissociation forming cob(II)alamin and hydroxyl radicals (∼1.5% yield) requires excitation to highly excited states. Dissociation is mediated by competition between internal conversion to the S1 surface and prompt bond cleavage.

Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate

The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates withdifferences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substratespecificityas SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C → 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.

Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase.

Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2″ of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a 5'-deoxyadenosyl 5'-radical (5'-dA•) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the ( S)-HPP and ( R)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA• generated by Fom3 abstracts the C2″- pro-R hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely ( S)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidylyl-2-fluoroethylphosphonate, which exhibits a lower C2″ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2″ radical, generated upon hydrogen atom abstraction by the 5'-dA•, directly attacks MeCbl to transfer a methyl radical (CH3•) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.

Characterization of the complex between native and reduced bovine serum albumin with aquacobalamin and evidence of dual tetrapyrrole binding.

Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (H2OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H2OCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior tothe formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the β-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H2OCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of H2OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.

Reaction kinetic studies for comparison of mutagenic potency between butadiene monoxide and glycidamide

DNA adducts can be formed from covalent binding of electrophilic reactive compounds to the nucleophilic N- and O-atoms of the biomolecule. The O-sites on DNA, with nucleophilic strength (n) of ca. 2, is recognized as a critical site for mutagenicity. Characterization of the reactivity of electrophilic compounds at the O-sites can be used to predict their mutagenic potency in relative terms. In the present study, reaction kinetic experiments were performed for butadiene monoxide (BM) in accordance with the Swain-Scott relation using model nucleophiles representing N- and O-sites on DNA, and earlier for glycidamide (GA) using a similar approach. The epoxide from the kinetic experiments was trapped by cob(I)alamin, resulting in formation of an alkylcobalamin which was analyzed by liquid chromatography tandem mass spectrometry. The Swain-Scott relationship was used to determine selectivity constant (s) of BM and GA as 0.86 and 1.0, respectively. The rate constant for the reaction at n of 2 was extrapolated to 0.023 and 0.038 M−1 h−1 for BM and GA, respectively, implying a higher mutagenic potency per dose unit of GA compared to BM. The reaction kinetic parameters associated with mutagenic potency were also estimated by a density functional theory approach, which were in accordance to the experimental determined values. These types of reaction kinetic measures could be useful in development of a chemical reactivity based prediction tool that could aid in reduction of animal experiments in cancer risk assessment procedures for relative mutagenicity.

Effects of medical food leucine content in the management of methylmalonic and propionic acidemias.

The current review highlights the varied effects of medical foods high in leucine (Leu) and devoid of valine (Val) and isoleucine (Ile) in the management of methylmalonic acidemia (MMA) and propionic acidemia and cobalamin C (cblC) deficiency, aiming to advance dietary practices. Leu is a key metabolic regulator with a multitude of effects on different organ systems. Recent observational studies have demonstrated that these effects can have unintended consequences in patients with MMA as a result of liberal use of medical foods. The combination of protein restriction and medical food use in MMA and propionic acidemia results in an imbalanced branched-chain amino acid (BCAA) dietary content with a high Leu-to-Val and/or Ile ratio. This leads to decreased plasma levels of Val and Ile and predicts impaired brain uptake of multiple essential amino acids. Decreased transport of methionine (Met) across the blood-brain barrier due to high circulating Leu levels is of particular concern in cblC deficiency in which endogenous Met synthesis is impaired. Investigations into the optimal composition of medical foods for MMA and propionic acidemia, and potential scenarios in which Leu supplementation may be beneficial are needed. Until then, MMA/propionic acidemia medical foods should be used judiciously in the dietary management of these patients and avoided altogether in cblC deficiency.

New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B12 non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblB.

Interaction between super-reduced cobalamin and selenite

The kinetics of the reaction between the two-electron reduced form of cobalamin (super-reduced cobalamin, cob(I)alamin, or Cbl(I)) and sodium selenite in an alkaline medium is studied spectrophotometrically. It is shown that the selenite rapidly oxidizes Cbl(I) to cob(II)alamin (Cbl(II)). It is established that the active form of the oxidant is the protonated selenite anion (HSeO3-), which receives six electrons during the reaction and transforms into HSe–. The reactions of cob(I)alamin oxidation by selenite and sulfite are compared.

Kinetic and Mechanistic Studies on the Reaction between Aquacobalamin and Selenocysteine

We studied the reaction between aquacobalamin (H2OCbl) and selenocysteine (Sec), a selenium‐containing analogue of cysteine, using conventional UV/Vis spectroscopy. This reaction leads to the formation of cob(II)alamin [Cbl(II)] without any detectable intermediate complexation of Cbl(III) with selenocysteine, whereas the interaction between H2OCbl and cysteine proceeds distinctly through the coordination of Cys onto the CoIII ion with further reduction of the CoIII–Cys complex to Cbl(II). The latter is slower than the reaction with Sec. We also showed that selenocystine is capable of accelerating the reduction of H2OCbl to Cbl(II) by cysteine.