Cobalamin Biosynthesis Research Articles

Crystal structure of Mycobacterium tuberculosis Rv2606c: A pyridoxal biosynthesis lyase

Tuberculosis is a lethal infectious disease caused by Mycobacterium tuberculosis. We determined the crystal structure of Rv2606c, a potential pyridoxal biosynthesis lyase (PdxS), from M. tuberculosis H37Rv at 1.8Å resolution. The overall structure of the protein, composed of a (β/α)8-barrel and two small 310-helices, was quite similar to those of other PdxS proteins. A glycerol molecule was observed to be bound at the active site of the Rv2606c structure through interactions with the conserved residues of Asp29 and Lys86, providing information regarding the potential active site and the substrate-binding environment of the protein. The interface for Rv2606c dodecamerization, which is primarily mediated by salt bridges and hydrophobic interactions, was quite different from those of other PdxS proteins. Furthermore, we observed that the Rv2606c and Rv2604c form a stable complex, suggesting that these proteins might function as PdxS and PdxT in M. tuberculosis.

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

The Vitamin B 6 Biosynthesis Pathway in Streptococcus pneumoniae Is Controlled by Pyridoxal 5′-Phosphate and the Transcription Factor PdxR and Has an Impact on Ear Infection

Vitamin B₆ is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5'-phosphate (PLP) biosynthesis pathway in Streptococcus pneumoniae. Our results revealed that S. pneumoniae possesses a de novo vitamin B₆ biosynthesis pathway encoded by the pdxST genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity in vitro. Deletion of pdxS, but not pdxT, resulted in a vitamin B₆ auxotrophic mutant. The defective growth of the ΔpdxS mutant in a vitamin B₆-depleted medium could be chemically restored in the presence of the B₆ vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of pdxS was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal ΔpdxR mutant also exhibited as a vitamin B₆ auxotroph. In addition, we found that disruption of the vitamin B₆ biosynthesis pathway in S. pneumoniae caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B₆ synthesis on virulence depends upon the bacterial infection niche.

Establishment of beet molasses as the fermentation substrate for industrial vitamin B12 production by Pseudomonas denitrificans

AbstractBACKGROUNDFermentation of the industrial vitamin B12 by Pseudomonas denitrificans usually utilizes sucrose or maltose as the sole carbon source, which results in increased medium costs. In order to decrease the fermentation cost, it is crucial and essential to employ a low‐cost and convenient raw material as an alternative medium substrate for industrial vitamin B12 production.RESULTSThe results obtained in chemically defined medium showed that glutamate and sucrose were favorable for cell growth and vitamin B12 biosynthesis of P. denitrificans. Due to containing a mass of ingredients such as sucrose, glutamate and betaine, beet molasses was consequently chosen as the main medium substrate for industrial P. denitrificans fermentation in a 120 000 L fermenter. Vitamin B12 production reached 181.75 mg L–1.CONCLUSIONSBeet molasses is a by‐product of the sugar industry and is thus very inexpensive, and it was proved that beet molasses was an efficient and economical medium substrate for industiral vitamin B12 fermentation by P. denitrificans. © 2013 Society of Chemical Industry

Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis

BackgroundIn the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8.ResultsThe primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum.ConclusionsThe conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology.

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B(12)) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH(60), that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH(60) was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH(60) demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.

Strategies for vitamin B6 biofortification of plants: a dual role as a micronutrient and a stress protectant

Vitamin B6 has an essential role in cells as a cofactor for several metabolic enzymes. It has also been shown to function as a potent antioxidant molecule. The recent elucidation of the vitamin B6 biosynthesis pathways in plants provides opportunities for characterizing their importance during developmental processes and exposure to stress. Humans and animals must acquire vitamin B6 with their diet, with plants being a major source, because they cannot biosynthesize it de novo. However, the abundance of the vitamin in the edible portions of the most commonly consumed plants is not sufficient to meet daily requirements. Genetic engineering has proven successful in increasing the vitamin B6 content in the model plant Arabidopsis. The added benefits associated with the enhanced vitamin B6 content, such as higher biomass and resistance to abiotic stress, suggest that increasing this essential micronutrient could be a valuable option to improve the nutritional quality and stress tolerance of crop plants. This review summarizes current achievements in vitamin B6 biofortification and considers strategies for increasing vitamin B6 levels in crop plants for human health and nutrition.

Co(ii)-detection does not follow Kco(ii) gradient: channelling in Co(ii)-sensing

The MerR-like transcriptional activator CoaR detects surplus Co(ll) to regulate Co(ll) efflux in a cyanobacterium. This organism also has cytosolic metal-sensors from three further families represented by Zn(ll)-sensors ZiaR and Zur plus Ni(ll)-sensor InrS. Here we discover by competition with Fura-2 that CoaR has KCo(ll) weaker than 7 × 10(-8) M, which is weaker than ZiaR, Zur and InrS (KCo(ll) = 6.94 ± 1.3 × 10(-10) M; 4.56 ± 0.16 × 10(-10) M; and 7.69 ± 1.1 × 10(-9) M respectively). KCo(ll) for CoaR is also weak in the CoaR-DNA adduct. Further, Co(ll) promotes DNA-dissociation by ZiaR and DNA-association by Zur in vitro in a manner analogous to Zn(ll), as monitored by fluorescence anisotropy. After 48 h exposure to maximum non-inhibitory [Co(ll)], CoaR responds in vivo yet the two Zn(ll)-sensors do not, despite their tighter KCo(ll) and despite Co(ll) triggering allostery in ZiaR and Zur in vitro. These data imply that the two Zn(ll) sensors fail to respond because they fail to gain access to Co(ll) under these conditions in vivo. Several lines of evidence suggest that CoaR is membrane associated via a domain with sequence similarity to precorrin isomerase, an enzyme of vitamin B12 biosynthesis. Moreover, site directed mutagenesis reveals that transcriptional activation requires CoaR residues that are predicted to form hydrogen bonds to a tetrapyrrole. The Co(ll)-requiring vitamin B12 biosynthetic pathway is also membrane associated suggesting putative mechanisms by which Co(ll)-containing tetrapyrroles and/or Co(ll) ions are channelled to CoaR.

Genotype-Specific Changes in Vitamin B6Content and the PDX Family in Potato

Vitamin B6 is one of the most versatile cofactors in plants and an essential phytonutrient in the human diet that benefits a variety of human health aspects. Although biosynthesis of the vitamin has been well resolved in recent years, the main research is currently based on Arabidopsis thaliana with very little work done on major crop plants. Here we provide the first report on interactions and expression profiles of PDX genes for vitamin B6 biosynthesis in potato and how vitamin B6 content varies in tubers of different genotypes. The results demonstrate that potato is an excellent resource for this vitamin and that strong natural variation in vitamin B6 content among the tested cultivars indicates high potential to fortify vitamin B6 nutrition in potato-based foods.

Effect of CoCl2 on the content of different metals and a relative activity of DNA‐hydrolyzing abzymes in the blood plasma of mice

Cobalt is a transition metal and an essential trace element that is required for vitamin B₁₂ biosynthesis, enzyme activation, and so on but is toxic in high concentrations. It was shown that the content of different elements in the plasma of 2-month-old BALB/c mice (control group) decreased in the following order: Ca > Mg > Si > Fe > Zn > Cu ≥ Al ≥ B. The treatment of mice with CoCl₂ did not appreciably change the relative content of Ca, Cu, and Zn, but a significant increase in the content of B (2.3-fold), Mg (1.5-fold), Al and Fe (2.1-fold), and Si (3.4-fold) was found. The treatment of mice led to a 2.2-fold decrease in the concentration of the total blood protein and a 1.7 ± 0.2-fold decrease of total immunoglobulin Gs (IgGs). Deoxyribonuclease IgGs corresponding to mice treated (t-IgGs) and non-treated (nt-IgGs) with CoCl₂ contained intrinsically bound metal ions; these IgGs hydrolyzed DNA with very low activity but were not active in the presence of ethylenediaminetetraacetic acid or after Ab dialysis against ethylenediaminetetraacetic acid. The average RAs of deoxyribonuclease nt-IgGs increased after addition of external metal ions in the following order: Zn²⁺< Ca²⁺ < Cu²⁺ < Fe²⁺ < Mn²⁺ < Mg²⁺ < Co²⁺ < Ni²⁺. Interestingly, t-IgGs demonstrated lower activities than those for nt-IgGs either in the absence of external metal ions (2.7-fold) or in the presence of Cu²⁺ (9.5-fold) > Co²⁺ (5.6-fold) > Zn²⁺ (5.1-fold) > Mg²⁺ (4.1-fold) > Ca²⁺ (3.0-fold) > Fe²⁺ (1.3-fold). However, the RAs of t-IgGs were remarkably more active than nt-IgGs in the presence of best activators of t-IgGs Ni²⁺ (1.4-fold) and especially Mn²⁺ (2.2-fold). The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions, and a change of metal-dependent specificity of Abzs.

Whole-genome sequencing and identification of Morganella morganii KT pathogenicity-related genes.

BackgroundThe opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria.ResultsThe 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes.ConclusionsThis is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis

The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B12) pathway in E. coli, and by His-tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly-bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Impact of lactobacilli on orally acquired listeriosis

Listeria monocytogenes is a foodborne pathogen that crosses the intestinal barrier and disseminates within the host. Here, we report a unique comprehensive analysis of the impact of two Lactobacillus species, Lactobacillus paracasei CNCM I-3689 and Lactobacillus casei BL23, on L. monocytogenes and orally acquired listeriosis in a gnotobiotic humanized mouse model. We first assessed the effect of treatment with each Lactobacillus on L. monocytogenes counts in host tissues and showed that each decreases L. monocytogenes systemic dissemination in orally inoculated mice. A whole genome intestinal transcriptomic analysis revealed that each Lactobacillus changes expression of a specific subset of genes during infection, with IFN-stimulated genes (ISGs) being the most affected by both lactobacilli. We also examined microRNA (miR) expression and showed that three miRs (miR-192, miR-200b, and miR-215) are repressed during L. monocytogenes infection. Treatment with each Lactobacillus increased miR-192 expression, whereas only L. casei association increased miR-200b and miR-215 expression. Finally, we showed that treatment with each Lactobacillus significantly reshaped the L. monocytogenes transcriptome and up-regulated transcription of L. monocytogenes genes encoding enzymes allowing utilization of intestinal carbon and nitrogen sources in particular genes involved in propanediol and ethanolamine catabolism and cobalamin biosynthesis. Altogether, these data reveal that the modulation of L. monocytogenes infection by treatment with lactobacilli correlates with a decrease in host gene expression, in particular ISGs, miR regulation, and a dramatic reshaping of L. monocytogenes transcriptome.

Effect of CoCl2 treatment on major and trace elements metabolism and protein concentration in mice

Cobalt (Co) is a transition metal and an essential trace element, required for vitamin B12 biosynthesis, enzyme activation and other biological processes, but toxic in high concentrations. There is lack of data for the effect of long-term Co(II) treatment on the concentrations of other trace elements. We estimate the influence of cobalt chloride (CoCl2) on the relative content of different metals in mouse plasma using two-jet arc plasmatron atomic emission and on the total protein content. On average, the content of different elements in the plasma of 2-month-old balb/c mice (control group) decreased in the order: Ca>Mg>Si>Fe>Zn>Cu≥Al≥B. The treatment of mice for 60days with CoCl2 (daily dose 125mg/kg) did not appreciably change the relative content of Ca, Cu, and Zn, while a 2.4-fold statistically significant decrease in the content of B and significant increase in the content of Mg (1.4-fold), Al and Fe (2.0-fold) and Si (3.2-fold) was found. A detectable amount of Mo was observed only for two control mice, while the plasma of 9 out of 16 mice of the treated group contained this metal. The administration of Co made its concentration detectable in the plasma of all mice of the treated group, but the relative content varied significantly. The treatment led to a 2.2-fold decrease in the concentration of the total plasma protein. Chronic exposure to CoCl2 affects homeostasis as well as the concentrations and metabolism of other essential elements, probably due to competition of Co ions for similar binding sites within cells, altered signal transduction and protein biosynthesis. Long-term treatment also leads to significant weight changes and reduces the total protein concentration.The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions and different physiological processes.

Changes in physiology and protein abundance in salt-stressed wheat chloroplasts

Leaves are the final site of salinity perception through the roots. To better understand how wheat chloroplasts proteins respond to salt stress, the study aimed to the physiochemical and comparative proteomics analysis. Seedlings (12-days-old) were exposed to 150 mM NaCl for 1, 2, or 3 days. Na(+) ions were rapid and excessively increase in roots, stems and leaves. Photosynthesis and transpiration rate, stomatal conductance, and relative water content decreased whereas the level of proline increased. Statistically significant positive correlations were found among the content of hydrogen peroxide, activity of catalase, and superoxide dismutase under salt stress in wheat. Protein abundance within the chloroplasts was examined by two-dimensional electrophoresis. More than 100 protein spots were reproducibly detected on each gel, 21 protein spots were differentially expressed during salt treatment. Using linear quadruple trap-Fourier transform ion cyclotron resonance (LTQ-FTICR) hybrid mass spectrometry, 65 unique proteins assigned in the differentially abundant spots. Most proteins were up-regulated at 2 and 3 days after being down-regulated at 1 day. Others showed only slight responses after 3 days of treatment, including Rubisco, glutamate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, photosystem I, and pyridoxal biosynthesis protein PDX1.2 and PDX1.3. The ATP synthase (α, β, and γ) and V-type proton ATPase subunits were down-regulated resulting showed negative impact by Na(+) on the photosynthetic machinery. This ephemeral increase and subsequent decrease in protein contents may demonstrate a counterbalancing influence of identified proteins. Several proteins such as cytochrome b6-f (Cyt b6-f), germin-like-protein, the γ-subunit of ATP synthase, glutamine synthetase, fructose-bisphosphate aldolase, S-adenosylmethionine synthase, carbonic anhydrase were gradually up-regulated during the period of treatment, which can be identified as marker proteins.

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae

BackgroundGeobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae.ResultsSequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53–56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration.ConclusionsGene acquisitions have expanded strain SZ’s respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

Study on the dissolved oxygen control strategy in large‐scale vitamin B12 fermentation by Pseudomonas denitrificans

AbstractBACKGROUND: There are two different routes for vitamin B12 biosynthesis, which results in discrepancies and uncertainties of the dissolved oxygen (DO) concentration for vitamin B12 fermentation. In this paper, the DO control strategy was explored for industrial vitamin B12 fermentation by Pesudomonas denitrificans in 120000‐L fermenter.RESULTS: A DO‐stat strategy was first successfully scaled up from a 9000 L fermenter to a 120 000 L fermenter. Then a multi‐stage DO control strategy was further established in the 120 000 L fermenter, in which the DO level was shifted from 8–10% (20–48 h) to 2–5% (49–106 h) and below 2% (107–168 h) by gradually reducing the rate of aeration and agitation. As a result, 198.80 mg L−1 of vitamin B12 was obtained, which was significantly higher than those obtained under the fermentations with one‐stage DO control.CONCLUSIONS: The comparatively low DO level was favorable for vitamin B12 biosynthesis, but it would have an extremely negative effect on cell growth. Compared with the low DO level maintained at all times of the fermentation process, a multi‐stage DO control strategy could not only increase the biomass but also improve vitamin B12 biosynthesis. Copyright © 2012 Society of Chemical Industry

Planetary change and biochemical adaptation: molecular evolution of corrinoid and heme biosyntheses

This review examines the evidence suggesting that the anaerobic biosynthesis of cobalamin (vitamin B12) evolved during early stages of cell evolution and was quickly recruited in the pathway leading to deoxyribonucleotides, the building blocks of DNA genomes. Biochemical evolution preceding the synthesis of the heme group and related molecules is discussed within the framework of geological evolution in which the appearance and accumulation of an oxygen-rich atmosphere stands as one of the major events in the evolution of the planet and the biosphere.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of pyridoxal biosynthesis lyase PdxS fromPyrococcus horikoshii

Pyridoxal biosynthesis lyase (PdxS) is an important player in the biosynthesis of pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B(6). PLP is an important cofactor involved in the metabolic pathway of amine-containing natural products such as amino acids and amino sugars. PdxS catalyzes the condensation of ribulose 5-phosphate (Ru5P), glyceraldehyde 3-phosphate (G3P) and ammonia, while glutamine amidotransferase (PdxT) catalyzes the production of ammonia from glutamine. PdxS and PdxT form a complex, PLP synthase, and widely exist in eubacteria, archaea, fungi and plants. To facilitate further structural comparisons among PdxS proteins, the structural analysis of PdxS from Pyrococcus horikoshii encoded by the Ph1355 gene was initiated. PdxS from P. horikoshii was overexpressed in Escherichia coli and crystallized at 296 K using 2-methyl-2,4-pentanediol as a precipitant. Crystals of P. horikoshii PdxS diffracted to 2.61 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 59.30, b = 178.56, c = 109.23 Å, β = 102.97°. The asymmetric unit contained six monomers, with a corresponding V(M) of 2.54 Å(3) Da(-1) and a solvent content of 51.5% by volume.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5'-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6 de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.