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Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39
FEBS Letters
Abstract
To clarify the physiological function of pseudovitamin B(12) (or adeninylcobamide; AdeCba) in Spirulina platensis NIES-39, cobalamin-dependent methionine synthase (MS) was characterized. We cloned the full-length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin-, zinc-, S-adenosylmethionine-, and homocysteine-binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.
Supplementary resource (1)
Nucleotide Sequence
March 2010
... Cobalamin is a complex molecule with a central cobalt-containing corrin ring, an α ligand of 5,6-dimethylbenzimidizole (DMB), and a β ligand of either OH-, CN-, Me-, or Ado-(12) (Fig. 1). Previous studies have shown that instead of producing cobalamin, Cyanobacteria produce pseudocobalamin (11,13,14), an analog of cobalamin in which adenine substitutes for DMB as the α ligand (12) (Fig. 1). Production of pseudocobalamin in a natural marine environment has not been shown, nor have reasons for the production of this compound in place of cobalamin been elucidated. ...
... SCM1, HCE1, HCA1, and PS0) also produced cobalamin (Table S1), confirming earlier suggestions based on the presence of cobalamin biosynthesis genes in Thaumarchaeota genomes (10). Like other Cyanobacteria (11,13,14), four axenic strains of marine Cyanobacteria (Prochlorococcus MED4 and MIT9313 and Synechococcus WH8102 and WH7803) produced pseudocobalamin (Table S1). In all of the cobalamin or pseudocobalamin producers, we detected compounds with β ligands Me-, Ado-, and OH-but not CN- (Table S1). ...
... The 3D structure of MetH contains three β pleated sheets and two α helices that form a pocket for the DMB ligand of cobalamin in Escherichia coli (31,32). Cyanobacterial MetH is predicted to form the same pocket (13). However, conserved amino acids within this pocket in the cyanobacterial MetH differ from sequences of organisms known to use cobalamin (Figs. ...
Significance
Cobalamin (vitamin B 12 )-dependent organisms span all domains of life, making procurement of the vitamin from the few prokaryotic producers an essential function in organismal interactions. Yet not all key producers of cobalamin have been identified in the ocean. We show that in the marine environment, select heterotrophic bacteria and Thaumarchaeota produce cobalamin, while Cyanobacteria, the most abundant phytoplankton on earth, supply and use pseudocobalamin. These chemically distinct cofactors support different members of the microbial community because they are not interchangeable as cofactors in enzymes. Our findings identify key organisms supporting cobalamin-based interdependencies that underpin primary production and microbial interactions in the ocean.
... The lower bases adenine in norpseudo-B 12 or DMB in DMB-Cba are uncoordinated and in the base-off conformation to anchor the cofactors deeply inside the RDase scaffold, suggesting that the lower base is not directly involved in catalysis (PDB ID = 4UR0, 4RAS). Interestingly, kinetic studies on non-RDase cobamide-dependent enzymes suggested varying affinities of the apoprotein to cobamides with different lower bases (Barker et al., 1960;Lengyel et al., 1960;Tanioka et al., 2010). For example, both the mammalian (sheep) kidney and bacterial (Propionibacterium shermanii) methylmalonyl-CoA mutase have high affinity to DMB-Cba (K m values of 0.021 and 0.024 μM, respectively), whereas the K m values for Bza-Cba were about an order in magnitude greater (Lengyel et al., 1960). ...
... In contrast, the glutamate mutase of Clostridium tetanomorphum preferred Bza-Cba over DMB-Cba with K m values of 0.24 and 18 μM, respectively (Barker et al., 1960). Preference for a cobamide with a specific lower base was also observed with the methionine synthase of Spirulina platensis strain NIES-39, which bound pseudo-B 12 with a K m of 0.07 μM, and a much higher K m of 16.0 μM was determined for DMB-Cba (Tanioka et al., 2010). These observations suggest that the affinity of the enzyme to cobamides with different lower bases affects the assembly of the functional holoenzyme, which can explain the observed lower base effects on reductive dechlorination rates and extents. ...
Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) μM and 67.4 (±1.4) μM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.The ISME Journal advance online publication, 10 November 2015; doi:10.1038/ismej.2015.197.
... Microalgae ingredients contain high levels of vitamin B12 although the origin of this compound is still an open debate. It is known that Spirulina is able to synthesize B12, but not in the active form while C. vulgaris does not synthesize at all this compound (Helliwell et al., 2016;Tanioka et al., 2010). Despite this, there are several studies that demonstrate the presence of active B12 in the dried biomass of these microalgae (Edelmann et al., 2019;Watanabe et al., 2013). ...
Energy bars are popular meal supplements due to their convenience and high nutritional content. Microalgae, such as Spirulina and Chlorella Vulgaris, are appealing food ingredients containing high-quality proteins and essential bioactive compounds. This study investigated the incorporation of these microalgae into a simple energy bar model at three levels of addition (0.0 %, 2.5% and 5.0%). Bars were characterized in terms of colour, water activity, moisture content, texture as well as nutritional and sensory profiles. Results showed that microalgae improved the protein and vitamin B12 content, and influenced color, flavor, and texture of the final product. Spirulina provided the most significant changes, increasing dark green colour, sea/fishy flavours and candies and grass tastes. Chlorella offered different colourways depending on the strain and brought to the sensory profile some umami/fishy notes that need to be taken into account in the formulation of commercial products.
... Vitamin B 12 is distinguished into its bioavailable form cobalamin and its biologically nonactive form pseudo-vitamin B 12 resulting from the α-ligand binding the cobalt in the center of the corrin ring [43]. Previous studies indicate primarily the enrichment of pseudovitamin B 12 in microalgae [44,45]. In the present study, the analyzed contents of bioactive vitamin B 12 in both microalgae were under 0.3 µg/100 g. ...
A 14-day randomized controlled study with a parallel design was conducted with 80 healthy participants. Intervention groups I (IG1) and II (IG2) received a defined background diet and consumed a smoothie enriched with either 15 g of Chlorella dry weight (d.w.) or 15 g of Microchloropsis d.w. daily. Control group II (CG2) received a defined background diet without the smoothie. Control group I (CG1) received neither. Blood samples and 24-h urine were collected at the beginning and the end of the study. Serum concentrations of 25-hydroxyvitamin D3, vitamin D3, selenium, iron, ferritin, transferrin saturation, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol and the LDL-cholesterol/HDL cholesterol ratio decreased in IG1 (p < 0.05), while 25-hydroxyvitamin D2 increased (p < 0.05). In IG2, vitamin D3, 25-hydroxyvitamins D2 and D3 decreased (p < 0.05), while concentrations of fatty acids C20:5n3 and C22:5n3 increased. Serum and urine uric acid increased in IG1 and IG2 (p < 0.05). Microchloropsis is a valuable source of n3 fatty acids, as is Chlorella of vitamin D2. Regular consumption of Chlorella may affect the iron and selenium status negatively but may impact blood lipids positively. An elevated uric acid concentration in blood and urine following the regular consumption of microalgae poses potential risks for human health.
... Cyanobacteria produce the non-bioavailable form, pseudocobalamin. which contains an adenine ring rather than 5,6 DMB at its base ( Fig. 1.7) (Helliwell et al., 2016;Tanioka et al., 2010;Watanabe et al., 1999;Taga and Walker, 2008). Pseudocobalamin actively competes with cobalamin for uptake by the intrinsic factor, which transports cobalamin around the blood (STUP-PERICH and NEXO, 1991). ...
Spirulina (Limnospira fusiformis) is an edible filamentous cyanobacterium with 'superfood' status. Spirulina powder can be used a food supplement, providing essential amino and polyunsaturated fatty acids, minerals and vitamins. In >25 clinical trials, Spirulina consumption has reduced the incidence of diabetes, hypertension, and improves immune system functions. Spirulina′s range of health applications, cheap minimal growth medium and fast growth rate compared to conventional crops make it an ideal candidate for supplementing astronaut diets and eventually providing long-term food supply if cultivated on Mars. However, despite being an attractive starting point for a range of applications, more traits could be added to enhance Spirulina uptake and usefulness further; including flavour improvement, enhancing its therapeutic abilities or nutritional enrichment. A major barrier to strain improvement is the lack of a routine system for Spirulina transformation. My project set out to 1: produce a system to enable precise changes to Spirulina′s genome and targeted introduction of novel genes, 2: add new useful traits to Spirulina utilising this technique. To address 1, a system utilising homologous recombination was developed and successfully demonstrated as a method for inserting transgenes into Spirulina. Antibiotic resistant gene ble and reporter mVenus showed the functionality of this approach. To address 2: genes mneI, encoding a super-sweet protein and pth, encoding osteoporosis drug Human Parathyroid Hormone, were introduced to produce organoleptic and therapeutic traits. Finally, testing Spirulina′s capabilities to be grown with minimal energy requirements will be essential to determine the practicality of using Spirulina strains for Earth and/or space applications. Therefore, the final objective was 3: investigate Spirulina cultivation with the resources available on a Mars base. Comprehensive Spirulina cultivation experiments using different lighting settings and media produced from Martian regolith and urine was explored. Spirulina could successfully grow on urine media, and does not require blue lighting for biomass production.
... Growth of the B. subtilis strain expressing P. megaterium metH in a medium lacking methionine was supported to various extents by most benzimidazolyl and both phenolyl cobamides, but not by Cba, the purinyl cobamides, or Cbi (Fig. 6D, red squares). This result indicates MetH-dependent growth is influenced by the corrinoid tail structure, as observed previously in other bacteria (78)(79)(80)(81)(82). In the B. subtilis strain containing the P. megaterium metE locus which includes the repressing SAM-Cbl-riboswitch, growth was suppressed by benzimidazolyl cobamides to different extents (Fig. 6D, blue circles). ...
In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found, among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semiselective. Analyses of chimeric riboswitches and structural models indicate, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites. IMPORTANCE Some bacterial mRNAs contain a region called a riboswitch which controls gene expression by binding to a metabolite in the cell. Typically, riboswitches sense and respond to a limited range of cellular metabolites, often just one type. In this work, we found the cobalamin (vitamin B12) riboswitch class is an exception, capable of sensing and responding to multiple variants of B12-collectively called corrinoids. We found cobalamin riboswitches vary in corrinoid specificity with some riboswitches responding to each of the corrinoids we tested, while others responding only to a subset of corrinoids. Our results suggest the latter class of riboswitches sense intrinsic conformational differences among corrinoids in order to support the corrinoid-specific needs of the cell. These findings provide insight into how bacteria sense and respond to an exceptionally diverse, often essential set of enzyme cofactors.
... Finally, some essential vitamins are missing from cyanobacteria, most notably the cobalamin vitamin B 12 (Helliwell et al., 2016;Miyamoto et al., 2006;Tanioka et al., 2010;Watanabe et al., 1999). Furthermore, these cyanobacteria synthesise a non-bioavailable version of cobalamin, termed pseudocobalamin, that can reduce the uptake of B 12 contained in other components of the diet (Stupperich and Nexo, 1991). ...
Establishing the first human presence on Mars will be the most technically challenging undertaking yet in the exploration beyond our planet. The remoteness of Mars from Earth, the inhospitable surface conditions including low atmospheric pressure and cold temperatures, and the need for basic resources including water, pose a formidable challenge to this endeavour. The intersection of multiple disciplines will be required to provide solutions for temporary and eventually permanent Martian habitation. This review considers the role cyanobacteria and eukaryotic microalgae (collectively referred to here as ‘microalgae’) may have in supporting missions to the red planet. The current research using these microorganisms in biological life support systems is discussed, with a systematic analysis of their usage in each system conducted. The potential of microalgae to provide astronauts with oxygen, food, bio-polymers and pharmaceuticals is considered. An overview of microalgal experiments in space missions across the last 60 years is presented, and the research exploring the technical challenges of cultivation on Mars is discussed. From these findings, an argument for culturing microalgae in subterranean bioreactors is proposed. Finally, future synthetic biology approaches for enhancing the cyanobacterial/microalgal role in supporting human deep-space exploration are presented. We show that microalgae hold significant promise for providing solutions to many problems faced by the first Martian settlers, however these can only be realised with significant infrastructure and a reliable power source.
... Lorsque la liaison de MetR à l'ADN est perturbée, l'activité de MetE est altérée 168 . Diverses études ont montré par ailleurs que la méthionine peut réprimer les niveaux d'expression des enzymes à différents niveaux dans sa voie de biosynthèse : sont ainsi impactées les enzymes impliquées dans l'activation de l'homosérine (MetX-HAT/HST), la production de cystathionine (les CGS), et la synthèse de l'homocystéine (via les CBL et les OAHS/OSHS) 95,124,169,170 Une variation mineure dans la formation de l'homocystéine existe cependant chez les cyanobactéries : plusieurs espèces de Synechocystis 172 , ainsi que Spirulina platensis 173 , utilisent en effet de l'adéninylcobamide (pseudo vitamine B12) comme cofacteur de la méthionine synthase MetH à la place de la cobalamine. Structurellement, l'adéninylcobamide diffère de la cobalamine par son groupement axial, qui est un fragment adéninyle, par opposition au motif ribonucléotyle 5,6diméthylbenzimidazole des MetH classiques (figure 33). ...
Acinetobacter baylyi ADP1 (ADP1), une bactérie du sol aux capacités de dégradation des molécules aromatiques remarquables est utilisée au laboratoire comme organisme modèle pour l’élucidation de nouvelles fonctions enzymatiques et voies métaboliques. Tirant profit de deux qualités d’ADP1 (compétence naturelle et recombinaison homologue efficace), une collection complète de mutants knock-out a été obtenue. L’analyse de cette ressource génomique a fait apparaître des phénotypes non prédits par l’annotation aboutissant notamment à l’initiation de l’étude du métabolisme du soufre chez ADP1 avec deux axes : (1) la biosynthèse de la L-méthionine (L-Met) et plus spécialement l’étude des familles d’enzymes non homologues MetX et MetA catalysant l’acylation de l’homosérine dans le monde vivant, première étape de cette voie ; (2) le recyclage de la L-méthionine et les voies d’assimilation des molécules soufrées. La thèse s’inscrit dans ces deux thématiques. Tout d’abord, un vaste projet combinant criblage expérimental et analyse structurale des sites actifs de MetA et MetX avait permis précédemment d’identifier les résidus déterminant l’usage de l’acyl-CoA et de proposer des règles précises de prédiction de fonction pour ces deux familles finalement isofonctionnelles. Cette étude avait révélé entre autres que 10% des MetX n’étaient pas impliqués dans la biosynthèse de L-Met. Nous avons donc entrepris la caractérisation de ces paralogues à l’activité inédite L-sérine O-succinyltransférase (SST) et finalement impliquées dans la biosynthèse de la L-cystéine (L-Cys). Jusqu'alors, l'acétylation par les L-sérine O-acétyltransférases (SAT) était le seul moyen connu d'activer la L-sérine. La détermination des paramètres cinétiques des SST ainsi que la caractérisation par LCMS in vitro puis la détection in vivo d’O-succinyl-L-sérine (OSS) dans les métabolomes de la levure Schizosaccharomyces pombe et la bactérie Xanthomonas campestris ont permis de démontrer la fonction de ces paralogues. Pour compléter la démonstration, la caractérisation des cystéine synthases (CysK) respectives a montré qu’elles effectuent en effet la sulfhydrylation de l’OSS pour former de la L-Cys. Notre étude a ainsi également révélé que la voie décrite de la biosynthèse de la L-cystéine chez les levures jusqu’alors extrapolée à partir de la levure modèle Saccharomyces cerevisae (voie de transsulfuration réverse) était en réalité une exception et que la grande majorité des levures synthétisent la L-Cys à partir de la L-sérine via ce nouveau métabolite, l’OSS. Cette thèse, dans un deuxième temps, a initié l’exploration expérimentale des voies d’assimilation du soufre chez ADP1 chez qui notamment aucune voie de recyclage de la L-Met n’était prédite alors qu’elle y est source de soufre. Combinant des approches complémentaires (génétique inverse par phénotypage de la collection complète de mutants sur diverses sources de soufre, transcriptomique sur L-Met et L-Cys versus sulfate, criblage des enzymes à PLP d’ADP1 potentiellement impliquées, étude biochimique des candidats et construction de mutants d’ADP1), une image assez précise des voies d’assimilation de diverses sources de soufre se dessine. Par exemple, les voies d’assimilations de la L-Met et du DMSP semblent aboutir à la production de sulfite via la synthèse de méthanesulfonate sous le contrôle du régulateur transcriptionnel CBL. De plus, le KMBA et le méthanethiol sont probablement des intermédiaires de la voie de recyclage de la L-Met, alors que le DMSO et le DMSO2 semblent être des intermédiaires cataboliques uniquement pour le DMSP.
... We applied VB 12 Path to identify and explore VB 12 biosynthesis gene families in the ocean and human intestinal environments. The taxonomic profiles are consistent with previous reports that Cyanobacteria (39)(40)(41), the most representative producer of pseudocobalamin, is the predominant group of cobalamin producers in ocean environment. Proteobacteria presents as the second most abundant group (23). ...
Cobalamin (vitamin B12; VB12) is an indispensable nutrient for all living entities in the Earth's biosphere and plays a vital role in both natural and host environments. Currently in the metagenomic era, gene families of interest are extracted and analyzed based on functional profiles by searching shotgun metagenomes against public databases. However, critical issues exist in applying public databases for specific processes such as VB12 biosynthesis pathways. We developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 biosynthesis gene families of microbial communities in complex environments. VB12Path contains a total of 60 VB12 synthesis gene families, 287,731 sequences, and 21,154 homology groups, and it aims to provide accurate functional and taxonomic profiles of VB12 synthesis pathways for shotgun metagenomes and minimize false-positive assignments. VB12Path was applied to characterize cobalamin biosynthesis gene families in human intestines and marine environments. The results demonstrated that ocean and human intestine had dramatically different VB12 synthesis processes and that gene families belonging to salvage and remodeling pathway dominated human intestine but were lowest in the ocean ecosystem. VB12Path is expected to be a useful tool to study cobalamin biosynthesis processes via shotgun metagenome sequencing in both environmental and human microbiome research. IMPORTANCE Vitamin B12 (VB12) is an indispensable nutrient for all living entities in the world but can only be synthesized by a small subset of prokaryotes. Therefore, this small subset of prokaryotes controls ecosystem stability and host health to some extent. However, critical accuracy and comprehensiveness issues exist in applying public databases to profile VB12 synthetic gene families and taxonomic groups in complex metagenomes. In this study, we developed a curated functional gene database termed VB12Path for accurate metagenomic profiling of VB12 communities in complex environments. VB12Path is expected to serve as a valuable tool to uncover the hidden microbial communities producing this precious nutrient on Earth.
... (Ade)CN-Cba was extracted from the cultured cells of Propionibacterium acidipropionici JCM6427 (RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan), purified to homogeneity, and identified according to the method described by Tanioka et al. [27]. (Ade)CN-Cba solution was bubbled with N 2 gas for 20 min, reduced with NaBH 4 , and neutralized with 1 M HCl. ...
Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.
... All cobamides share the same catalytic features, which include an upper ligand (R in Figure 1B) that varies for different chemical reactions; for the MMUT-catalyzed reaction, the upper ligand is 5'-deoxyadenosine (as in adenosylcobalamin, AdoCbl), while for MS it is a methyl group (as in methylcobalamin, MeCbl). Although the lower ligand is not directly involved in catalysis, lower ligand structure affects the biochemistry of cobamide-dependent enzymes, including orthologs of both MMUT and MS (17)(18)(19)(20)(21)(22)(23)(24). However, the differential effects of cobamides have been primarily studied in bacterial cobamide-dependent enzymes, and only to a limited extent in mammalian MS and MMUT. ...
Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds – cobamides – that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.
... The human gut commensal bacterium Bacteroides thetaiotaomicron could use benzimidazolyl and purinyl cobamides for MetH-dependent growth but could not use phenolyl cobamides (28). An example of MetH selectivity in vitro was in Spirulina platensis, where the purified enzyme bound its native cobamide, pseudocobalamin, with a higher affinity than for cobalamin (72). An exception to this observed selectivity is another gut pathogen, Salmonella enterica, which can use its native cobamide, pseudocobalamin, in addition to cobalamin, [Phe]Cba, and [Cre]Cba, for MetH-dependent growth, although other cobamides were not tested (48,49). ...
The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B 12 ) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile -associated disease.
... In a subset of base-off enzymes, referred to as "base-off/His-on," a histidine residue from the protein coordinates the cobalt ion in place of the lower ligand (61,63). Despite its distance from the reactive center, lower ligand structure affects the activity of base-off enzymes, as evidenced by the cobamide cofactor selectivity of methionine synthase (71), methylmalonyl coenzyme A (CoA) mutase (MCM) (60,72), reductive dehalogenases (49), and other enzymes (59,72,73). However, the mechanisms by which lower ligand structure affects the biochemistry of base-off cobamidedependent enzymes remain unclear. ...
Cobamides, including vitamin B 12 , are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti , largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.
... The vast majority of cyanobacteria, and thus also Spirulina, synthesise and utilise pseudovitamin B12 as a cofactor for specialised methionine synthase (METH; EC 2.1.1.14), a structure that prefers an adenine instead of DMBI as a lower ligand in the B12 structure (Helliwell et al., 2016;Tanioka et al., 2010). Pseudovitamin B12 exhibits a ca. ...
This study aimed to investigate the riboflavin, niacin, folate and B12 content in microalgae powders. Riboflavin was determined with an ultra-high-performance liquid chromatographic (UHPLC) method after extraction and a two-enzyme treatment. Niacin analysis involved mild acidic hydrolysis, with niacin quantitated as the sum of nicotinic acid and nicotinamide using a UHPLC method. Both a microbiological (MBA) and a UHPLC method was used for vitamin B12 quantification as a cyanocobalamin (CNCbl) and UHPLC-mass spectrometry was used to confirm the vitamin B12 form. Total folate was determined both with MBA and as a sum of the vitamers with a UHPLC method after extraction and tri-enzyme treatment.
The riboflavin and niacin content varied from 21 to 41 μg/g and 0.13–0.28 mg/g, respectively, in Chlorella sp., Spirulina (Arthrospira sp.) and Nannochloropsis gaditana powders. Chlorella powders were, on average, richer in total folate (19.7 μg/g) than Spirulina powders were (3.5 μg/g). The sum of the folate vitamers determined with UHPLC matched better with the microbiological total folate content in Chlorella than in Spirulina powders. Pseudovitamin B12 was the predominant form over active vitamin B12 in Spirulina powders, whereas Chlorella sp. and N. gaditana powders solely contained active vitamin B12 up to 2.1 μg/g.
... In a subset of base-off enzymes, referred to as "base-off/His-on," a histidine residue from the protein coordinates the cobalt ion in place of the lower ligand ( Drennan et al., 1994;Mancia et al., 1996). Despite its distance from the reactive center, lower ligand structure affects the activity of base-off enzymes, as evidenced by the cobamide cofactor selectivity of methionine synthase ( Tanioka et al., 2010), methylmalonyl-CoA mutase (MCM) ( Lengyel et al., 1960;Poppe et al., 1997), reductive dehalogenases ( Keller et al., 2018), and other enzymes ( Barker et al., 1960;Lengyel et al., 1960;Poppe et al., 2000). However, the mechanisms by which lower ligand structure affects the biochemistry of base-off cobamidedependent enzymes remain unclear. ...
Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B 12 , are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria.
... Corrinoid-dependent enzymes function in the utilization of propanediol, ethanolamine and other carbon and nitrogen sources; degradation of certain amino acids, odd chain fatty acids and cholesterols; biosynthesis of methionine, deoxynucleotides and antibiotics; tRNA modification; mercury methylation; acetogenesis; methanogenesis; and halogenated solvent degradation ( Matthews 2009). r Corrinoids with different lower ligands are not functionally equivalent; an organism can only use a subset of the corrinoids that may be present in its environment ( Tanioka et al. 2010;Yi et al. 2012;Mok and Taga 2013). ...
Microbial communities govern numerous fundamental processes on earth. Discovering and tracking molecular interactions among microbes is critical for understanding how single species and complex communities impact their associated host or natural environment. While recent technological developments in DNA sequencing and functional imaging have led to new and deeper levels of understanding, we are limited now by our inability to predict and interpret the intricate relationships and interspecies dependencies within these communities. In this review, we highlight the multifaceted approaches investigators have taken within their areas of research to decode interspecies molecular interactions that occur between microbes. Understanding these principles can give us greater insight into ecological interactions in natural environments and within synthetic consortia.
... and valine in S. platensis and they sequenced the genes ilvX and ilvW encoding these two enzymes. Tanioka et al. (2010) characterized cobalamin-dependent methionine synthase to study the physiological function of pseudovitamin B12 or adeninylcobamide in Spirulina platensis NIES-39 and finally cloned the full-length Spirulina MS. Linjawi (2011) investigated the protective effect of Spirulina against mitomycin C (MMC)-induced genotoxic damage in male rats and suggested that Spirulina exerts its anti-mutagenic properties by inhibiting alterations in the gene expression. ...
Cyanobacteria are prokaryotes which can perform photosynthesis like higher plants. Their genomic organisation is very simple and thus is suitable for the study of detailed photosynthesis mechanism at a molecular level also for many other genomic manipulations relevant to benefit of living organisms. This unicellular alga, Spirulina has a thin thread like elongated structure and classified under Cyanobacteriaceae which is blue green in colour. Under microscope it looks like bunch of bright helical threads (Fig. 11.1).
... Our results indicated that because naturally grown N. flagelliforme contain substantial amounts of pseudo B 12 , which is inactive in humans [7] and because the fake items have very low B 12 contents, commercially available hair vegetable is not suitable for use of B 12 source, regardless of the presence of the fake items. Cyanobacteria have the ability to synthesize pseudo B 12 [13], which functions as a coenzyme of methionine synthase to catalyze the synthesis of methionine from homocysteine and N 5 -methyltetrahydrofolate [14]. In the present study, the cultured Nostoc sample I predominantly contained B 12 but not pseudo B 12 ( Table 2), suggesting that N. flagelliforme may synthesize both B 12 and pseudo B 12 de novo. ...
Vitamin B 12 contents in the edible cyanobacterium Nostoc flagelliforme, also known as hair vegetable, were as-sayed using a microbiological method. We detected high vitamin B 12 contents in samples of naturally grown cells (109.2 ± 18.5 μg/100g dry weight) and cultured cells (120.2 ± 53.6 μg/100g dry weight). However, commercially available hair vegetable samples, which comprised fake substitutes and Nostoc, had variable contents (4.8 -101.6 μg/100g dry weight) because concomitant fake items contain very low vitamin B 12 contents. To evaluate whether natural and cultured N. flagelliforme samples contained vitamin B 12 or pseudovitamin B 12 , corrinoid compounds were purified and identified as pseudovitamin B 12 (approximately 72%) and vitamin B 12 (approximately 28%) using silica gel 60 TLC bioautography and LC/MS. The results suggested that N. flagelliforme contains substan-tial amounts of pseudovitamin B 12 , which is inactive in humans.
... For example, the K m of the methylmalonyl coenzyme A (CoA) mutase enzyme of Propionibacterium freudenreichii subsp. shermanii, glutamate mutase of Clostridium tetanomorphum, and methionine synthase of Arthrobacter platensis are influenced by the structure of the lower ligand (5)(6)(7). In addition, coordination of the lower ligand to the central cobalt ion can affect the reactivity of the cofactor (8,9). ...
Phenolyl cobamides are unique members of a class of cobalt-containing cofactors that includes vitamin B12 (cobalamin). Cobamide cofactors facilitate diverse reactions in prokaryotes and eukaryotes. Phenolyl cobamides are structurally
and chemically distinct from the more commonly used benzimidazolyl cobamides such as cobalamin, as the lower axial ligand
is a phenolic group rather than a benzimidazole. The functional significance of this difference is not well understood. Here
we show that in the bacterium Sporomusa ovata, the only organism known to synthesize phenolyl cobamides, several cobamide-dependent acetogenic metabolisms have a requirement
or preference for phenolyl cobamides. The addition of benzimidazoles to S. ovata cultures results in a decrease in growth rate when grown on methanol, 3,4-dimethoxybenzoate, H2 plus CO2, or betaine. Suppression of native p-cresolyl cobamide synthesis and production of benzimidazolyl cobamides occur upon the addition of benzimidazoles, indicating
that benzimidazolyl cobamides are not functionally equivalent to the phenolyl cobamide cofactors produced by S. ovata. We further show that S. ovata is capable of incorporating other phenolic compounds into cobamides that function in methanol metabolism. These results demonstrate
that S. ovata can incorporate a wide range of compounds as cobamide lower ligands, despite its preference for phenolyl cobamides in the
metabolism of certain energy substrates. To our knowledge, S. ovata is unique among cobamide-dependent organisms in its preferential utilization of phenolyl cobamides.
Cobalamin influences marine microbial communities because an exogenous source is required by most eukaryotic phytoplankton, and demand can exceed supply. Pseudocobalamin is a cobalamin analogue produced and used by most cyanobacteria but is not directly available to eukaryotic phytoplankton. Some microbes can remodel pseudocobalamin into cobalamin, but a scarcity of pseudocobalamin measurements impedes our ability to evaluate its importance for marine cobalamin production. Here, we perform simultaneous measurements of pseudocobalamin and methionine synthase (MetH), the key protein that uses it as a co‐factor, in Synechococcus cultures and communities. In Synechococcus sp. WH8102, pseudocobalamin quota decreases in low temperature (17°C) and low nitrogen to phosphorus ratio, while MetH did not. Pseudocobalamin and MetH quotas were influenced by culture methods and growth phase. Despite the variability present in cultures, we found a comparably consistent quota of 300 ± 100 pseudocobalamin molecules per cyanobacterial cell in the Northwest Atlantic Ocean, suggesting that cyanobacterial cell counts may be sufficient to estimate pseudocobalamin inventories in this region. This work offers insights into cellular pseudocobalamin metabolism, environmental and physiological conditions that may influence it, and provides environmental measurements to further our understanding of when and how pseudocobalamin can influence marine microbial communities.
Bacteria encounter chemically similar nutrients in their environment that impact their growth in distinct ways. Among such nutrients are cobamides, the structurally diverse family of cofactors related to vitamin B 12 (cobalamin), which function as cofactors for diverse metabolic processes. Given that different environments contain varying abundances of different cobamides, bacteria are likely to encounter cobamides that enable them to grow robustly as well as those that do not function efficiently for their metabolism. Here, we performed a laboratory evolution of a cobamide-dependent strain of Escherichia coli with pseudocobalamin (pCbl), a cobamide that E. coli uses less effectively than cobalamin for MetH-dependent methionine synthesis, to identify genetic adaptations that lead to improved growth with less-preferred cobamides. After propagating and sequencing nine independent lines and validating the results by constructing targeted mutations, we found that mutations that increase expression of the outer membrane cobamide transporter BtuB are beneficial during growth under cobamide-limiting conditions. Unexpectedly, we also found that overexpression of the cobamide adenosyltransferase BtuR confers a specific growth advantage in pCbl. Characterization of the latter phenotype revealed that BtuR and adenosylated cobamides contribute to optimal MetH-dependent growth. Together, these findings improve our understanding of how bacteria expand their cobamide-dependent metabolic potential.
The microbiological assay of total cobalamin (vitamin B12) by Lactobacillus delbrueckii subsp. lactis ATCC7830 is now used worldwide in food analysis because of its high sensitivity, low running cost, and no expensive instruments. It has been recently reported that some foods contain a substantial number of inactive corrinoid compounds, some of which are active in this bacterium. These results indicate that the microbiological method must be replaced with high-performance liquid chromatography or liquid chromatography/electrospray ionization-tandem mass spectrometry as there can specifically determine biologically active cobalamin. Nowadays, powerful tools, such as immunoaffinity columns, purify cobalamin simply and specifically. In this chapter, we summarized the determination methods of cobalamin and related compounds in foods. Various inactive corrinoids found in foods were also characterized.
In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found that among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semi-selective. Analyses of chimeric riboswitches and structural models indicate that, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites.
In this study, we determined the vitamin B12 content of commercially-available edible insect products using a bioassay based on Lactobacillus delbrueckii ATCC 7830. Although the vitamin content of giant water bug, bee larva, grasshopper, and weaver ant products was low, we found that diving beetle and cricket products contained relatively high amounts of vitamin B12 (approximately 89.5 and 65.8 µg/100 g dry weight, respectively). In the cricket products most widely circulated as foods, specific corrinoid (vitamin B12) compounds were extracted and identified using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Despite the bioassay detecting high vitamin B12 content (approximately 50–75 µg/100 g dry weight) in these cricket products, UPLC–MS/MS analysis indicated that pseudovitamin B12 and 2-methylmercaptoadenyl cobamide (also known as factor S) were actually the predominant corrinoid compounds (∼74% and ∼21%, respectively), with authentic vitamin B12 making up only 5% of total corrinoids.
The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.
IMPORTANCE
Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.
Nutritional interdependencies
Bacteria and archaea show a wide range of nutritional specialism. Not every organism can synthesize essential components and may need to trade for them. Taking as an example a diverse and interesting family of enzyme cofactors—the cobalt-containing cobamides, which include vitamin B 12 —Sokolovskaya et al. reviewed the interdependencies among microorganisms for this suite of nutrients. Cobamides are required for many processes, from catabolism of carbon sources to nucleotide biosynthesis, and are needed by a majority of microbes, from those in the gut to those in the oceans. Availability of cobamides is patchy and habitat specific, and nonspecific scavenging may not be adequate to obtain a specific cobamide structure required by an organism. Therefore, a variety of mutualisms have evolved to deliver and import specific structural variants of cobamides between organisms or among consortia of eukaryotes and prokaryotes by an equal variety of subtle and distinct mechanisms.
Science , this issue p. 48
Nearly all microbes rely on other species in their environment to provide nutrients they are unable to produce. Nutritional interactions include not only the exchange of carbon and nitrogen compounds, but also amino acids and cofactors. Interactions involving cross-feeding of cobamides, the vitamin B12 family of cofactors, have been developed as a model for nutritional interactions across species and environments. In addition to experimental studies, new developments in culture-independent methodologies such as genomics and modeling now enable the prediction of nutritional interactions in a broad range of organisms including those that cannot be cultured in the laboratory. New insights into the mechanisms and evolution of microbial nutritional interactions are beginning to emerge by combining experimental, genomic, and modeling approaches.
Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B 12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent metabolisms encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile , we studied C. difficile 630 Δ erm and mutant derivatives under cobamide-dependent conditions in vitro . Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis, and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δ erm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinc acid or the late intermediate cobinamide, and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δ erm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in in the btuFCD locus of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.
Importance
The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B 12 ) metabolism of C. difficile has been underexplored, though it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo , but produces them when given cobamide precursors. Better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile- associated disease.
Vitamin B12 is synthesized by only certain bacteria and archaea but not by animals or plants. In marine environments, bacterial vitamin B12 is transferred and concentrated into fish and shellfish bodies by plankton in the marine food chain. Moreover, marine macrophytic red algae, Porphyra spp. specifically contain substantial amounts of vitamin B12, due to microbial interaction. Although some meats or viscera of edible fish and shellfish are excellent sources of biologically active vitamin B12, an inactive corrinoid, pseudovitamin B12, was found in some edible shellfish using liquid chromatography/electrospray ionization–tandem mass spectrometry. To prevent elderly people from developing vitamin B12 deficiency due to food protein-bound vitamin B12 malabsorption, we present a survey of marine foods containing free vitamin B12. The results of our study suggest that bonito and clam extracts (or soup stocks), which contain considerable amounts of free vitamin B12 are useful not only as seasonings and flavorings but also as excellent sources of free vitamin B12.
Over the past two decades, biotechnologies have provided a motor for innovation and sustainability in many economies all around the world by developing new processes and products in a bio-economy approach. Besides food and feed, increasing interest on biomass derived fuels, chemicals and materials, sustainably sourced and produced, has raised, providing an alternative to heavy reliance on finite fossil fuel resources. One of the most innovative and promising sectors of the bio-economy is related to bio-based products, obtained in part or entirely from organic biomass, which account for about 16% of world production of bio-economy’s products. Plant biomass is rich in high added value compounds; mainly antioxidants and fibres, which once extracted can serve as green fine chemicals or can be used in food supplements and/or nutraceutical sector.
A great deal of evidence has established that the secondary compounds of higher plants (i.e. polyphenols) inhibit and/or quench free radicals and reactive oxygen species (ROS) thus protecting against oxidative damage. These compounds can therefore be exploited as additives in a large number of different commodities, such as plastics and nanomaterials.
This chapter gives an insight into the relevant research results regarding the valorization of polyphenol fractions extracted from agricultural wastes, focusing on those derived from fruit production and transformation. Structure-activity relationships will be discussed in view of their use in the field of innovative materials.
Vitamin B12 was determined and characterized in 19 dried Chlorella health supplements. Vitamin contents of dried Chlorella cells varied from < 0.1 μg to approximately 415 μg per 100 g dry weight. Subsequent liquid chromatography/electrospray ionization-tandem mass spectrometry analyses showed the presence of inactive corrinoid compounds, a cobalt-free corrinoid, and 5-methoxybenzimidazolyl cyanocobamide (factor IIIm) in four and three high vitamin B12-containing Chlorella tablets, respectively. In four Chlorella tablet types with high and moderate vitamin B12 contents, the coenzyme forms of vitamin B12 5'-deoxyadenosylcobalamin (approximately 32%) and methylcobalamin (approximately 8%) were considerably present, whereas the unnaturally occurring corrinoid cyanocobalamin was present at the lowest concentrations. The species Chlorella sorokiniana (formerly C. pyrenoidosa) is commonly used in dietary supplements and did not show an absolute requirement of vitamin B12 for growth despite vitamin B12 uptake from the medium being observed. In further experiments, vitamin B12-dependent methylmalonyl-CoA mutase and methionine synthase activities were detected in cell homogenates. In particular, methionine synthase activity was significantly increased following the addition of vitamin B12 to the medium. These results suggest that vitamin B12 contents of Chlorella tablets reflect the presence of vitamin B12 generating organic ingredients in the medium or the concomitant growth of vitamin B12-synthesizing bacteria under open culture conditions.
Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B12-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B12, suggesting that the intrinsic pseudovitamin B12 served as a substitute of coenzyme B12. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.
The microbial mechanisms and key metabolites that shape the composition of the human gut microbiota are largely unknown, impeding efforts to manipulate dysbiotic microbial communities toward stability and health. Vitamins, which by definition are not synthesized in sufficient quantities by the host and can mediate funda- mental biological processes in microbes, represent an attractive target for reshaping microbial communities. Here, we discuss how vitamin B12 (cobalamin) impacts diverse host-microbe symbioses. Although cobalamin is synthesized by some human gut microbes, it is a precious resource in the gut and is likely not provisioned to the host in significant quantities. However, this vitamin may make an unrecognized contribution in shaping the structure and function of human gut microbial communities.
Cyanobacteria produce numerous bioactive compounds, most of which have therapeutic properties. Substantial amounts of cyanobacterial cells are thus produced worldwide to meet the high demand of both the food and pharmaceutical industries. Cyanobacteria contain substantial amounts of phycobiliproteins, which act as light-harvesting accessory pigments. C-Phycocyanin (blue pigment protein), in particular, has been reported as a potent peroxy radical scavenger, platelet aggregation inhibitor, and proliferation inhibitor of cancer cells in vitro and in vivo. The therapeutic activity of the blue pigment protein may be due to the phycobilin (an open tetrapyrrole compound) of the molecule. Early ecological studies suggested that certain cyanobacteria play a role as producers of vitamin B12 (a corrinoid) in nature. Vitamin B12 (a closed tetrapyrrole compound), which is the largest (molecular weight 1355.4) and most complex of all the vitamins, is synthesized only in certain bacteria and then concentrated mainly in the bodies of higher predatory organisms in the natural food chain system. Although large amounts of vitamin B12 are determined in cyanobacteria (blue-green algae), there has been considerable controversy concerning the bioavailability of the algal vitamin B12 in humans. Here, we reviewed up-to-date information on de novo synthesis pathway, characterization, and physiological functions of corrinoids and phycobilins as the closed and open tetrapyrroles, respectively, in cyanobacteria.
Methionine is essential in all organisms, as it is both a proteinogenic amino acid and a component of the cofactor, S-adenosyl methionine. The metabolic pathway for its biosynthesis has been extensively characterized in Escherichia coli; however, it is becoming apparent that most bacterial species do not use the E. coli pathway. Instead, studies on other organisms and genome sequencing data are uncovering significant diversity in the enzymes and metabolic intermediates that are used for methionine biosynthesis. In this review, we have summarized the different biochemical strategies that are employed in the three key steps for methionine biosynthesis from homoserine (i.e. acylation, sulfurylation and methylation). We have surveyed the presence and absence of the various biosynthetic enzymes in 1,593 representative bacterial species, shedding light on the non-canonical nature of the E. coli pathway. We have also highlighted ways in which knowledge of methionine biosynthesis can be utilized for biotechnological applications. Finally, we have noted gaps in the current understanding of bacterial methionine biosynthesis. For example, we discuss the presence of one gene (metC) in a large number of species that appear to lack the gene encoding the enzyme for the preceding step in the pathway (metB), as it is understood in E. coli. Therefore, this review aims to move the focus away from E. coli, in order to better reflect the true diversity of bacterial pathways for methionine biosynthesis.
High-performance thin-layer chromatography (HPTLC) is a separation technique commonly used to identify and quantify compounds in chemical mixtures. Preliminary experiments indicated that Lichrospher silica gel 60 F254s HPTLC sheets were the most suitable for analyzing vitamin B12 compounds. This study revealed the advantages of miniaturized Lichrospher HPTLC for analyzing authentic vitamin B12 compounds as short migration distances (5 cm) and short development times (<45 min) in combination with high separation efficiency and sensitivity (>25 pmol at 254 nm). The practicability of using miniaturized HPTLC was demonstrated by the separation and identification of vitamin B12 compounds purified from foods using an immunoaffinity column.
Cobamides such as vitamin B12 (cobalamin) are produced exclusively by prokaryotes and used by many other organisms as cofactors for diverse metabolic processes. Cobamides are cobalt-containing tetrapyrroles with upper and lower axial ligands. The structure of the lower ligand varies in cobamides produced by different bacteria. We investigated the biochemical basis of this structural variability by exploring the reactivity of homologs of CobT, the enzyme responsible for activating lower ligand bases for incorporation into cobamides. Our results show that CobT enzymes can activate a range of lower ligand substrates, and the majority of the enzymes tested preferentially attach 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin. This suggests that many bacteria that synthesize cobamides other than cobalamin in pure culture may produce cobalamin in mixed communities by attaching DMB when it is available in the environment.
Vitamin B(12) content of an edible cyanobacterium, Nostochopsis sp. was determined to be 140.6±16.2 μg/100 g dry weight by a microbiological method. To evaluate whether the Nostochopsis cells contain vitamin B(12) or inactive corrinoid compounds, corrinoid compounds were purified from the cells and then identified as pseudovitamin B(12) (97.4±11.8 μg/100 g dry weight) and vitamin B(12) (43.2±6.0 μg/100 g dry weight) on the basis of silica gel 60 TLC bioautograms and LC/ESI-MS/MS chromatograms. Vitamin B(12) content was significantly increased in the Nostochopsis cells (254.8±17.6 μg/100 g dry weight) grown in the vitamin B(12)-supplemented medium.
The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli
was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle
lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann
fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is
coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the
protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent
enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will
be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757
and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine
synthase.
This review examines deoxyadenosylcobalamin (Ado-B12) biosynthesis, transport, use, and uneven distribution among living forms. We describe how genetic analysis of enteric bacteria has contributed to these issues. Two pathways for corrin ring formation have been found-an aerobic pathway (in P. denitrificans) and an anaerobic pathway (in P. shermanii and S. typhimurium)-that differ in the point of cobalt insertion. Analysis of B12 transport in E. coli reveals two systems: one (with two proteins) for the outer membrane, and one (with three proteins) for the inner membrane. To account for the uneven distribution of B12 in living forms, we suggest that the B12 synthetic pathway may have evolved to allow anaerobic fermentation of small molecules in the absence of an external electron acceptor. Later, evolution of the pathway produced siroheme, (allowing use of inorganic electron acceptors), chlorophyll (O2 production), and heme (aerobic respiration). As oxygen became a larger part of the atmosphere, many organisms lost fermentative functions and retained dependence on newer, B12 functions that did not involve fermentation. Paradoxically, Salmonella spp. synthesize B12 only anaerobically but can use B12 (for degradation of ethanolamine and propanediol) only with oxygen. Genetic analysis of the operons for these degradative functions indicate that anaerobic degradation is important. Recent results suggest that B12 can be synthesized and used during anaerobic respiration using tetrathionate (but not nitrate or fumarate) as an electron acceptor. The branch of enteric taxa from which Salmonella spp. and E. coli evolved appears to have lost the ability to synthesize B12 and the ability to use it in propanediol and glycerol degradation. Salmonella spp., but not E. coli, have acquired by horizontal transfer the ability to synthesize B12 and degrade propanediol. The acquired ability to degrade propanediol provides the selective force that maintains B12 synthesis in this group.
Vitamin B12 (cobalamin) was identified nearly 80 years ago as the anti-pernicious anaemia factor in liver, and its importance in human health and disease has resulted in much work on its uptake, cellular transport and utilization. Plants do not contain cobalamin because they have no cobalamin-dependent enzymes. Deficiencies are therefore common in strict vegetarians, and in the elderly, who are susceptible to an autoimmune disorder that prevents its efficient uptake. In contrast, many algae are rich in vitamin B12, with some species, such as Porphyra yezoensis (Nori), containing as much cobalamin as liver. Despite this, the role of the cofactor in algal metabolism remains unknown, as does the source of the vitamin for these organisms. A survey of 326 algal species revealed that 171 species require exogenous vitamin B12 for growth, implying that more than half of the algal kingdom are cobalamin auxotrophs. Here we show that the role of vitamin B12 in algal metabolism is primarily as a cofactor for vitamin B12-dependent methionine synthase, and that cobalamin auxotrophy has arisen numerous times throughout evolution, probably owing to the loss of the vitamin B12-independent form of the enzyme. The source of cobalamin seems to be bacteria, indicating an important and unsuspected symbiosis.
Corrinoid (vitamin B12-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing
an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions,
S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B12, but it can make B12 from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added
DMB converted Cbi to pseudo-B12 cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install
adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B12 cofactors ceases and B12 cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type
cells make pseudo-B12 cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B12 for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B12 cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B12. Models are described for the mechanism and control of α-axial ligand installation.
Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.
• cobalamin-dependent methionine synthase
• cobalamin-independent methionine synthase
• methyl transferases
• zinc enzymes
• zinc inversion
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
In this study, we have been able to demonstrate the production and liberation of vitamin B12 (up to 16.5 pg/ml after 31 days of growth) by a blue green alga, Anabaena flos-aquae, in non-axenic and axenic cultures. This finding has a relevant ecological significance since it shows that, like bacteria, blue green algae may play a role as producers of vitamin B12.
To clarify the physiological function of pseudovitamin B(12) in cyanobacteria, we determined pseudovitamin B(12) contents and cobalamin-dependent methionine synthase activity in Synechocystis sp. PCC6803 grown under CoSO(4)-sufficient and -limited conditions. Pseudovitamin B(12) and cobalamin-dependent methionine synthase activity (0.8 nmol/min/mg protein) were found in a homogenate of the Synechocystis cells grown for 10 d in the CoSO(4)-sufficient medium. The cellular pseudovitamin B(12 )contents increased significantly at the early logarithmic growth phase and thereafter decreased rapidly at the stationary phase; a similar fluctuation pattern was shown in the cobalamin-dependent methionine synthase activity. Although the CoSO(4)-limited conditions did not reduce the cell growth, pseudovitamin B(12) contents and methionine synthase activity decreased significantly in the limited cells relative to the sufficient cells. These results indicate that the cyanobacterium Synechocystis sp. PCC6803 can synthesize pseudovitamin B(12) de novo and utilize it as the cofactor for cobalamin-dependent methionine synthase.
In this study, we have been able to demonstrate the production and liberation of vitamin B12 (up to 16.5 pg/ml after 31 days of growth) by a blue green alga. Anabaena flos-aquae, in non-axenic and axenic cultures. This finding has a relevant ecological significance since it shows that, like bacteria, blue green algae may play a role as producers of vitamin B12.
An incubation system containing rat jejunoileum was used to determine whether analogues of cyanocabalamin (CNCbl) inhibited the in vitro absorption of CNCbl. The naturally occurring analogues "pseudo B12," "factor A," and the nonnaturally occurring analogue desdemethyl B12, were produced by guided biosynthesis with Propionibacterium arabinosum. Factor B was produced by acid cleavage of cyanocabalamin. Inhibition of Co57-CNBbl uptake was demonstrated for cold CNCbl and for desdemethyl B12, although statistical significance was achieved only for cold CNCbl at 40X and 100X the molar quantity of the radiolabeled CNCbl. However, a trend towards progressive inhibition of specific CNCbl binding was shown as increasing concentrations of CNCbl or desdemethyl B12 were used. No inhibition of specific CNCbl binding could be demonstrated for any of the naturally occurring cobamides tested.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Background:
In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase.
Results:
We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions.
Conclusions:
The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.
Methionine synthase (MetH) catalyzes the transfer of a methyl group from bound methylcobalamin to homocysteine, yielding enzyme-bound cob(I)alamin and methionine. The cofactor is then remethylated by methyltetrahydrofolate. We now demonstrate that MetH is able to catalyze methylation of free cob(I)alamin with methyltetrahydrofolate. MetH had previously been shown to catalyze methylation of homocysteine with free methylcobalamin as the methyl donor, in a reaction that is first-order in added methylcobalamin, and we have confirmed this observation using homogenous enzyme. A truncated polypeptide lacking the cobalamin-binding region of the holoenzyme, MetH(2-649), was overexpressed and purified to homogeneity. MetH(2-649) catalyzes the methylation of free cob(I)alamin by methyltetrahydrofolate and the methylation of homocysteine by free methylcobalamin. Furthermore, a protein comprising residues 2-353 of the holoenzyme has now been overexpressed and purified to homogeneity, and this protein catalyzes methyl transfer from free methylcobalamin to homocysteine but not from methyltetrahydrofolate to free cob(I)alamin. The mutations Cys310Ala and Cys311Ala in MetH(2-649) completely abolish methyl transfer from exogenous methylcobalamin to homocysteine but do not affect methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, consistent with a modular construction for MetH. We infer that MetH is a modular protein comprising four separate regions: a homocysteine binding region (residues 2-353), a methyltetrahydrofolate binding region (residues 354-649), a region responsible for binding the cobalamin prosthetic group (residues 650-896), and an AdoMet-binding domain (residues 897-1227).
Effect of C-phycocyanin (from Spirulina platensis) pretreatment on carbontetrachloride and R-(+)-pulegone-induced hepatotoxicity in rats was studied. Intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of phycocyanin to rats, one or three hours prior to R-(+)-pulegone (250 mg/kg) or carbontetrachloride (0.6 ml/kg) challenge, significantly reduced the hepatotoxicity caused by these chemicals. For instance, serum glutamate pyruvate transaminase (SGPT) activity was almost equal to control values. The losses of microsomal cytochrome P450, glucose-6-phosphatase and aminopyrine-N-demethylase were significantly reduced, suggesting that phycocyanin provides protection to liver enzymes. It was noticed that the level of menthofuran, the proximate toxin of R-(+)-pulegone was nearly 70% more in the urine samples collected from rats treated with R-(+)-pulegone alone than rats treated with the combination of phycocyanin and R-(+)-pulegone. The possible mechanism involved in the hepatoprotection is discussed.
The vitamin B(12) concentration of an algal health food, spirulina (Spirulina sp.) tablets, was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor chemiluminescence methods. The values determined with the microbiological method were approximately 6-9-fold greater in the spirulina tablets than the values determined with the chemiluminescence method. Although most of the vitamin B(12) determined with the microbiological method was derived from various vitamin B(12) substitutive compounds and/or inactive vitamin B(12) analogues, the spirulina contained a small amount of vitamin B(12) active in the binding of the intrinsic factor. Two intrinsic factor active vitamin B(12) analogues (major and minor) were purified from the spirulina tablets and partially characterized. The major (83%) and minor (17%) analogues were identified as pseudovitamin B(12) and vitamin B(12), respectively, as judged from data of TLC, reversed-phase HPLC, (1)H NMR spectroscopy, ultraviolet-visible spectroscopy, and biological activity using L. leichmannii as a test organism and the binding of vitamin B(12) to the intrinsic factor.
Spirulina platensis NIES-39 was grown under open culture system in the presence or absence of CoSO(4) (12 microg/L) and/or vitamin B(12) (10 microg/L) to confirm whether CoSO(4) and/or vitamin B(12) stimulate or are essential for growth of the algal cells and for accumulation of vitamin B(12). The addition of CoSO(4) and/or vitamin B(12) could not affect both cell growth and cell yield of the alga. The amount of corrinoid-compound was increased significantly by the addition of CoSO(4) but not by vitamin B(12). A C18 reversed-phase HPLC pattern of the Spirulina corrinoid-compound increased by the addition of CoSO(4) was identical to that of authentic pseudovitamin B(12), which is inactive for human. These results indicate that the algal cells grown in the absence of CoSO(4) are suitable for use of human health foods because the inactive corrinoid-compound can be reduced significantly.
The biotechnology of microalgae has gained considerable importance in recent decades. Applications range from simple biomass production for food and feed to valuable products for ecological applications. For most of these applications, the market is still developing and the biotechnological use of microalgae will extend into new areas. Considering the enormous biodiversity of microalgae and recent developments in genetic engineering, this group of organisms represents one of the most promising sources for new products and applications. With the development of sophisticated culture and screening techniques, microalgal biotechnology can already meet the high demands of both the food and pharmaceutical industries.
The vitamin B12 concentration of the dried cells of Aphanizomenon flos-aquae was determined by both microbiological method with Lactobacillus delbrueckeii ATCC7830 and chemiluminescence method with intrinsic factor. The Aphanizomenon cells contained 616.3 +/- 30.3 micro g (n = 4) of vitamin B12 per 100 g of the dried cells by the microbiological method. The values determined with the chemiluminescence method, however, were only about 5.3% of the values determined by the microbiological method. A corrinoid-compound was purified from the dried cells and characterized. The purified corrinoid-compound was identified as pseudovitamin B12 (an inactive corrinoid-compound for humans) by silica gel 60 TLC, C18 reversed-phase HPLC, ultraviolet-visible spectroscopy, and 1H NMR spectroscopy. The results suggest that the Aphanizomenon cells are not suitable for use as a vitamin B12 source, especially in vegans.
The usual dietary sources of vitamin B(12) are animal foods, meat, milk, egg, fish, and shellfish. As the intrinsic factor-mediated intestinal absorption system is estimated to be saturated at about 1.5-2.0 microg per meal under physiologic conditions, vitamin B(12) bioavailability significantly decreases with increasing intake of vitamin B(12) per meal. The bioavailability of vitamin B(12) in healthy humans from fish meat, sheep meat, and chicken meat averaged 42%, 56%-89%, and 61%-66%, respectively. Vitamin B(12) in eggs seems to be poorly absorbed (< 9%) relative to other animal food products. In the Dietary Reference Intakes in the United States and Japan, it is assumed that 50% of dietary vitamin B(12) is absorbed by healthy adults with normal gastro-intestinal function. Some plant foods, dried green and purple lavers (nori) contain substantial amounts of vitamin B(12), although other edible algae contained none or only traces of vitamin B(12). Most of the edible blue-green algae (cyanobacteria) used for human supplements predominantly contain pseudovitamin B(12), which is inactive in humans. The edible cyanobacteria are not suitable for use as vitamin B(12) sources, especially in vegans. Fortified breakfast cereals are a particularly valuable source of vitamin B(12) for vegans and elderly people. Production of some vitamin B(12)-enriched vegetables is also being devised.
Small cells dominate photosynthetic biomass and primary production in many marine ecosystems. Traditionally, picoplankton refers to cells < or =2 microm. Here we extend the size range of the organisms considered to 3 microm, a threshold often used operationally in field studies. While the prokaryotic component of picophytoplankton is dominated by two genera, Prochlorococcus and Synechococcus, the eukaryotic fraction is much more diverse. Since the discovery of the ubiquitous Micromonas pusilla in the early 1950s, just over 70 species that can be <3 microm have been described. In fact, most algal classes contain such species. Less than a decade ago, culture-independent approaches (in particular, cloning and sequencing, denaturing gradient gel electrophoresis, FISH) have demonstrated that the diversity of eukaryotic picoplankton is much more extensive than could be assumed from described taxa alone. These approaches revealed the importance of certain classes such as the Prasinophyceae but also unearthed novel divisions such as the recently described picobiliphytes. In the last couple of years, the first genomes of photosynthetic picoplankton have become available, providing key information on their physiological capabilities. In this paper, we discuss the range of methods that can be used to assess small phytoplankton diversity, present the species described to date, review the existing molecular data obtained on field populations, and end up by looking at the promises offered by genomics.
Purification and characterization of a corrinoidcompound in an edible Cyanobacterium Aphanizomenon flos-aquae as a nutritional supplementary food
- E Miyamoto
- Y Tanioka
- T Nakao
- F Balra
- H Inui
- T Fujita
- F Watanabe
- Y Nakano
Miyamoto, E., Tanioka, Y., Nakao, T., Balra, F., Inui, H., Fujita, T., Watanabe, F.
and Nakano, Y. (2006) Purification and characterization of a corrinoidcompound in an edible Cyanobacterium Aphanizomenon flos-aquae as a
nutritional supplementary food. J. Agric. Food Chem. 54, 9604-9607.