Adenosylcobalamin Research Articles

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20–10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5–20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in elimination of all the fluorides with formation of acetaldehyde.

3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical centre compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

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.

Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9.

The introduction of complex biosynthetic pathways into the hosts' chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5-10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L-1 coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts' genomes, thus facilitating pathway optimization for chemical production.

The structure-activity relationship of Vitamin B12 and cobalamins and their nutritional correlations

This article reviews the so-called “cobalamins”, with special emphasis on coenzyme B12, discussing aspects of its structure-function relationship as well as various implications in biochemical, physiological and nutritional areas. Despite being one of the micronutrients with the lowest presence in organisms, cobalt (Co) is a constituent part of highly relevant enzymatic cofactors and associated with various biochemical and/or enzymatic processes. Vitamin B12 really represents a focus of interest of a multi- and interdisciplinary nature, since it is a metallic complex, an organometallic compound (for example, in the methylcobalamin form we identify the Cobalt-Carbon (Co-C) bond) and a group of relevance biophysics, as it interacts with different proteins in the biological environment, in addition to its impactful nutritional role, the deficiency of which causes the so-called “pernicious anemia”. This literature review article aims to offer a general and interdisciplinary overview of the topic, covering relevant topics in cobalt chemistry, mainly its coordination chemistry, which have been considered as model systems both for the study of metallic complexes properly considered and for the evaluation of the structure- metalloprotein activity.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

The coenzyme B12 precursor 5,6-dimethylbenzimidazole is a flavin antagonist in Salmonella.

Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB12) de novo only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB12 in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in S. Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report in vitro and in vivo evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in S. Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of S. Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the cycA (encodes D-Ala/D-Ser transporter) and dctA (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.

A synthetic cell-free 36-enzyme reaction system for vitamin B12 production

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

Coordination Chemistry Controls Coenzyme B12 Synthesis by Human Adenosine Triphosphate:Cob(I)alamin Adenosyltransferase.

Cobalamin (or vitamin B12)-dependent enzymes and trafficking chaperones exploit redox-linked coordination chemistry to control the cofactor reactivity during catalysis and translocation. As the cobalt oxidation state decreases from 3+ to 1+, the preferred cobalamin geometry changes from six- to four-coordinate (4-c). In this study, we reveal the sizable thermodynamic gain that accrues for human adenosine triphosphate (ATP):cob(I)alamin adenosyltransferase (or MMAB) by enforcing an unfavorable 4-c cob(II)alamin geometry. MMAB-bound cob(II)alamin is reduced to the supernucleophilic cob(I)alamin intermediate during the synthesis of 5'-deoxyadenosylcobalamin. Herein, we report the first experimentally determined reduction potential for 4-c cob(II)alamin (-325 ± 9 mV), which is 180 mV more positive than for the five-coordinate (5-c) water-liganded species. The redox potential of MMAB-bound cob(II)alamin is within the range of adrenodoxin, which we demonstrate functions as an electron donor. We also show that stabilization of 5-c cob(II)alamin by a subset of MMAB patient variants compromises the reduction by adrenodoxin, explaining the underlying pathogenic mechanism.

Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B12 Catalysis.

Adenosylcobalamin (AdoCbl), or coenzyme B12 , is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co-C bond and conformational stabilization of the adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

Process optimization of metabolically engineered Escherichia coliNSK015 fermentation for progressive improvement of 1,3‐propanediol production

AbstractBACKGROUNDPreviously, Escherichia coli NSK015 was developed for high yield of 1,3‐propanediol (1,3‐PDO) production. To further improve 1,3‐PDO concentration, parameters including kLa values with different agitations and impeller numbers, concentrations of coenzyme B12 and feeding strategies during fed‐batch fermentation were investigated.RESULTSIn this study, aerobic conditions at 300 rpm agitation and 1 vvm aeration with two Rushton turbine impellers (kLa = 33.6 h−1) and the concentration of coenzyme B12 at 7.5 μmol L−1 were identified as the best optimized conditions to improve 1,3‐PDO production by the strain. With a two‐pulsed continuous feeding, E. coli NSK015 produced 1,3‐PDO up to 60.3 g L−1 with the 1,3‐PDO yield approaching a theoretical maximum of 0.97 g g−1 and productivity of 0.42 g L−1 h−1 in fed‐batch fermentation, in which concentration was improved about 60% compared to that of batch fermentation.CONCLUSIONThe result indicated the efficiency of E. coli NSK015 in producing 1,3‐PDO under optimal aerobic conditions. The strain could even enhance growth and maintain enzymatic activities involved in the 1,3‐PDO pathway without utilizing antibiotics, isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) or enriching nutrients. Plasmid instability, high production cost related to medium preparation and purification, and waste disposal were not of concern. This may provide a new insight for large‐scale 1,3‐PDO production by E. coli NSK015. Additionally, E. coli NSK015 could be a microbial host model for further developing new 1,3‐PDO‐producing microorganisms regardless of plasmid, inducer, antibiotics and rich nutrients in fermentation medium. © 2023 Society of Chemical Industry (SCI).

Bivalent molecular mimicry by ADP protects metal redox state and promotes coenzyme B12 repair

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine(dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

Tuning of B12 photochemistry in the CarH photoreceptor to avoid radical photoproducts.

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Abstract 2200: The impact of patient mutations on coenzyme B12 loading and repair

Abstract 2200: The impact of patient mutations on coenzyme B12 loading and repair

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected byfood or other environmentalconditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012-fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.