Cobalamin Biosynthesis Research Articles

Global transcriptional response of Lactobacillus reuteri to the sourdough environment

Lactobacillus reuteri is a lactic acid bacterium that is highly adapted to the sourdough environment. It is a dominant member of industrial type II sourdoughs, and is also able to colonize the intestinal tract of mammals, including humans, and birds. In this study, the transcriptional response of L. reuteri ATCC 55730 was investigated during sourdough fermentation by using whole-genome microarrays. Significant changes of mRNA levels were found for 101 genes involved in diverse cellular processes, such as carbohydrate and energy metabolism, cell envelope biosynthesis, exopolysaccharide production, stress responses, signal transduction and cobalamin biosynthesis. The results showed extensive changes of the organism's gene expression during growth in sourdough as compared with growth in chemically defined medium, and, thus, revealed pathways involved in the adaptation of L. reuteri to the ecological niche of sourdough. The utilization of starch and non-starch carbohydrates, the remodelling of the cell wall, characterized by reduced d-alanylation, and increased amounts of cell wall-associated polysaccharides, as well as the regulatory function of two component systems for cell wall biogenesis and metabolism were suggested by the gene expression data as being important for growth in sourdough. The impact of several L. reuteri genes for effective growth in sourdough was shown by implementation of mutant strains in sourdough fermentation. This study contributes to the understanding of the molecular fundamentals of L. reuteri's ecological competitiveness, and provides a basis for further exploration of genetic traits involved in adaptation to the food environment.

Mutation in the cobO gene generates auxotrophy for cobalamin and methionine and impairs the symbiotic properties of Sinorhizobium fredii HH103 with soybean and other legumes

We report here the isolation of a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 obtained by Tn5-lacZ mutagenesis. Sequence analysis showed that the transposon was inserted into a gene homologous to cobO. This gene codes for a cobalamin adenosyltransferase which is involved in the biosynthesis of vitamin B12. Another HH103 cobO mutant (strain SVQ524), was constructed by the insertion of Omega interposon. Both cobO mutants required the addition of methionine because cobalamin acts as a cofactor of the enzyme MetH, which catalyses the last step of the methionine biosynthesis. Mutant SVQ524 failed to nodulate on Vigna radiate but was able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan, although the total number of nodules formed was highly reduced in comparison with that of plants inoculated with the wild-type strain HH103. The roots of these plants did not seem to secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. In all cases, the phenotype of SVQ524 was nearly overcome by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene in cosmid pMUS756.

Analysis of a Horizontally Transferred Pathway Involved in Vitamin B6 Biosynthesis from the Soybean Cyst Nematode Heterodera glycines

Heterodera glycines is an obligate plant parasite capable of biochemically and developmentally altering its host's cells in order to create a specialized feeding cell. Although the exact mechanism of feeding cell morphogenesis remains a mystery, the nematode's ability to manipulate the plant is thought to be due in part to horizontal gene transfers (HGTs). A bioinformatic screen of the nematode genome has revealed homologues of the genes SNZ and SNO, which comprise a metabolic pathway for the de novo biosynthesis of pyridoxal 5'-phosphate, the active form of vitamin B(6) (VB(6)). Analysis of the 2 genes, HgSNZ and HgSNO, show that they contain nematode-like introns, generate polyadenylated mRNAs, and map to the soybean cyst nematode genetic linkage map, indicating that they are part of the nematode genome. However, gene synteny, protein homology, and phylogenetic evidence suggest prokaryotic origin. This would represent the first case of the HGT of a complete pathway into a nematode or terrestrial animal. VB(6) acts as a cofactor in over 140 different enzymes, and recent studies point toward an important role as a potent quencher of reactive oxygen species. With H. glycines' penchant for acquiring parasitism genes through HGT along with the absence of this pathway in other land-based animals suggests a specific need for VB(6) which may involve the parasite-host interaction.

Influence of Zn2+, Co2+ and dimethylbenzimidazole on vitamin B12 biosynthesis by Pseudomonas denitrificans

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B12 biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn2+, Co2+ and DMBI on vitamin B12 production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B12. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B12 production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.

Optimizing vitamin B 12 biosynthesis by mycelial cultures of Lentinula edodes (Berk.) Pegl.

Preparations derived from Lentinula edodes (Berk.) Pegl. are widely used as dietary supplements, they contain compounds with immune system enhancing and chemopreventive properties. These preparations contain a blend of minerals and vitamins, including vitamins A, D, B 1, B 2, C, and niacin, but only scant vitamin B 12. Our goal was to optimize the growth conditions of submerged mycelial cultures of L. edodes in order to obtain a new dietary supplement enriched in vitamin B 12. We designed a biotechnological process in which cobalamin precursors, cobalt chloride and the methyl donors betaine, methionine, and choline, were added to the culture medium. The vitamin B 12 content in mycelial extracts was determined by RP-HPLC, while total Co 2+ content was determined by HPIC. At the optimal Co 2+ concentration (40 μg/mL), the vitamin B 12 level in L. edodes mycelium reached 95 μg/g of dry weight. All methyl donors had a positive effect on cobalamin biosynthesis, at least doubling its concentration in mycelia as compared mycelia grown in non-enriched medium. The amount of cobalamin in submerged cultivated mycelia of L. edodes was 10,000-fold higher than that recorded for fruiting bodies. Our results suggest that these optimized culture conditions could be applied to obtain a new cobalamin-enriched dietary supplement derived from L. edodes.

Comparative Genome Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112T and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112T has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B12). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with 13C3-glycerol demonstrated that L. reuteri JCM 1112T could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112T to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Crystal structure of a PduO‐type ATP:cobalamin adenosyltransferase from Burkholderia thailandensis

Crystal structure of a PduO‐type ATP:cobalamin adenosyltransferase from <i>Burkholderia thailandensis</i>

Two Distinct Roles for Two Functional Cobaltochelatases (CbiK) in Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Vitamin B(12), the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6A resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Proteomics Approach for Investigating the Disease Resistance Using Cucumber as Model Plant

The profile of proteins induced in cucumber leaves pretreated with Acibenzolar-S-Methyl (ASM) and challenge inoculated with Colletotrichum orbiculare are presented. ASM was applied to the first leaf and the third leaf collected at different time intervals of 0-48 h and the total protein extracted using trichloroacetic acid acetone was analyzed by two-dimensional gel electrophoresis. Increased expressions of pyridoxine biosynthesis protein, carbonic anhydrase, proteasome subunit (beta type 1), phosphomannomutase and ascorbate peroxidase proteins were noticed in cucumber plants systemically induced resistance by ASM. All the above proteins are well known to play a major role in scavenging of active oxygen species and preventing the cell from oxidative damage. Our results suggest that production and scavenging of ROS are one of the major events that occur during ASM induced systemic resistance in cucumber.

Cloning of the Pyridoxine 5′-Phosphate Phosphatase Gene (pdxP) and Vitamin B6Production inpdxPRecombinantSinorhizobium meliloti

A novel gene (pdxP) encoding a pyridoxine 5'-phosphate (PNP) phosphatase involved in the last step of pyridoxine biosynthesis was cloned from Sinorhizobium meliloti IFO 14782 on the basis of the peptide sequences of the natural enzyme. The pdxP gene is an open reading frame (708 bp) encoding 235 amino acid residues with a calculated molecular weight of 26,466. From its deduced amino acid sequence, it was predicted that the enzyme belongs to the haloacid dehalogenase superfamily. Transformants of Escherichia coli and S. meliloti by pdxP gene expression plasmids showed stimulated PNP phosphatase activities. When pdxP was overexpressed together with the PNP synthase gene (pdxJ) in S. meliloti, the recombinant strain produced 149 mg/l of pyridoxine, 46% and 16% higher than the host strain and the pdxJ recombinant of S. meliloti respectively.

Trapping of a Chromophoric Intermediate in the Pdx1‐Catalyzed Biosynthesis of Pyridoxal 5′‐Phosphate

I320 has it: The serendipitous trapping, by aromatization, of an enzyme-bound chromophoric intermediate (I320; see picture, blue box) involved in vitamin B6 formation using a common biological reducing agent provides new information regarding the structure of the chromophoric species.

Trapping of a Chromophoric Intermediate in the Pdx1‐Catalyzed Biosynthesis of Pyridoxal 5′‐Phosphate

Trapping of a Chromophoric Intermediate in the Pdx1‐Catalyzed Biosynthesis of Pyridoxal 5′‐Phosphate

The Pdx1 family is structurally and functionally conserved between Arabidopsis thaliana and Ginkgo biloba

Vitamin B6 is one of the most important compounds in living organisms, and its biosynthesis has only recently been understood. Because it is required for more than 100 biochemical reactions, lack of the vitamin is fatal. This is of special importance to mammals and humans, which cannot biosynthesize the vitamin and thus depend on its external uptake. Here we describe the cloning of a vitamin B6 biosynthetic gene GbPDX1 from Ginkgo biloba. The gene is expressed in seeds, leaf and trunk tissue. Using yeast 2-hybrid and pull-down assays, we show that the protein can interact with itself and with members of Arabidopsis thaliana AtPDX1 and AtPDX2 families. Furthermore, we prove the function of GbPDX1 in vitamin B6 biosynthesis by complementation of an Arabidopsis AtPDX1.3 mutant rsr4-1, at the phenotypical level and increasing vitamin B6 levels caused by ectopic GbPDX1 expression in the mutant background. Overall, this study provides a first description of Ginkgo vitamin B6 metabolism, and demonstrates a high degree of conservation between Ginkgo and Arabidopsis.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Biosynthesis and Use of Cobalamin (B12).

This review summarizes research performed over the last 23 years on the genetics, enzyme structures and functions, and regulation of the expression of the genes encoding functions involved in adenosylcobalamin (AdoCbl, or coenzyme B12) biosynthesis. It also discusses the role of coenzyme B12 in the physiology of Salmonella enterica serovar Typhimurium LT2 and Escherichia coli. John Roth's seminal contributions to the field of coenzyme B12 biosynthesis research brought the power of classical and molecular genetic, biochemical, and structural approaches to bear on the extremely challenging problem of dissecting the steps of what has turned out to be one of the most complex biosynthetic pathways known. In E. coli and serovar Typhimurium, uro'gen III represents the first branch point in the pathway, where the routes for cobalamin and siroheme synthesis diverge from that for heme synthesis. The cobalamin biosynthetic pathway in P. denitrificans was the first to be elucidated, but it was soon realized that there are at least two routes for cobalamin biosynthesis, representing aerobic and anaerobic variations. The expression of the AdoCbl biosynthetic operon is complex and is modulated at different levels. At the transcriptional level, a sensor response regulator protein activates the transcription of the operon in response to 1,2-Pdl in the environment. Serovar Typhimurium and E. coli use ethanolamine as a source of carbon, nitrogen, and energy. In addition, and unlike E. coli, serovar Typhimurium can also grow on 1,2-Pdl as the sole source of carbon and energy.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Genomic Differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, Identified by Suppression Subtractive Hybridization

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

Vitamin B1 biosynthesis in plants requires the essential iron–sulfur cluster protein, THIC

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3'-untranslated region of THIC. Strong evidence is provided for the involvement of an iron-sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.

Filling the gap of intracellular dephosphorylation in the Plasmodium falciparum vitamin B1 biosynthesis

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo. TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5′-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.