Bioconversion of Dethiobiotin into Biotin by Resting Cells and Protoplasts of Bacillus sphaericus bioB Transformant

The reaction system for the bioconversion of dethiobiotin into biotin by resting cells and protoplasts of a Bacillus sphaericus bioB transformant was established. The reaction mixtures consisted of completely synthetic components, such as amino acids and metal salts. Among the sulfur compounds tested, L-CyS and L-cystine were effective in the biosynthesis of biotin from dethiobiotin both by resting cells and by protoplasts. The optimum concentrations of L-Cys were 2 to 3 mM and more than 0.25 mM for resting cell and protoplast systems, respectively. Vigorous shaking enhanced the biotin biosynthesis by protoplasts. The addition of yeast extract to the reaction mixture without a mixture of amino acids brought about a three-fold increase in the, amount of biotin synthesized by protoplasts when compard to the case with the reaction mixture containing the amino acid mixture. The amount of biotin synthesized by protoplasts increased with the incubation time up to 6 h and reached about 2 μg/ml. There was a clear correlation between the number of remaining protopiasts and their biotin-biosynthesizing activity during the incubation.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Discussion of the occurrence of an intermediate hydroxylation.

The last step of the biosynthesis of biotin, i.e. the conversion of dethiobiotin to biotin was studied using E. coli. The three dethiobiotin derivatives hydroxylated at C-2 or C-5 were synthesized and tested as potential precursors of biotin. It appears that none of these compounds is able to support the growth of E. coli C124, a mutant which does not synthesize dethiobiotin, but converts it into biotin. These results strongly disfavour the hypothesis of the activation of the saturated carbons by an hydroxylation process.     

Adenine cycle in hepatopancreocytes of Helix pomatia (Gastropoda)

Intact hepatopancreocytes were obtained from hibernating or active purinotelic snails, H. pomatia (Gastropoda). When incubated with [14C]glycine or [14C]formate, they synthesized de novo purine compounds, including also adenylates, adenosine and adenine. Hepatopancreocytes resynthesized also adenylates and other purine compounds from [3H]adenine or from [3H]adenosine split by the H. pomatia cell enzyme to adenine; the resynthesis of ADP+ATP was proportional to adenine concentration. Thus all reactions of the postulated adenine cycle: AMP leads to adenosine leads to adenine leads to AMP occur in the intact hepatopancreocytes; this cycle could probably be responsible for maintenance of the high level of adenylates during winter sleep.     

Action of 5-(2-thienyl) valeric acid as a biotin antagonist

5-(2-Thienyl)valeric acid (TVA), a biotin analogue which can be easily prepared through chemical process, inhibited the growth of a biotin synthesizing Rhodotorula glutinis. The growth inhibition was reversed by the addition of biotin. Among biotin intermediates, dethiobiotin and 7,8-diaminopelargonic acid reversed the inhibition by TVA, while 7-keto-8-amino-pelargonic acid and pimelic acid did not. From these results, it was concluded that TVA is a biotin antagonist which probably acts as an inhibitor of biotin biosynthesis.     

A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis

Cofactor biosynthetic pathways represent a rich source of potential antibiotic targets. The second step in biotin biosynthesis is performed by BioA, a pyridoxal 5′-phosphate (PLP)-dependent enzyme. This enzyme has been confirmed as a candidate target in Mycobacterium tuberculosis; however, the current bioassay used to measure BioA activity is cumbersome and low throughput. Here we describe the design, development, and optimization of a continuous coupled fluorescence displacement assay to measure BioA activity. In this coupled assay, BioD converts the product of the BioA-catalyzed reaction into dethiobiotin, which is subsequently detected by displacement of a fluorescently labeled dethiobiotin probe from streptavidin. The assay was further adapted to a high-throughput screening format and validated against the LOPAC¹²⁸⁰ library.     

Antitumor Activity of a Pterin Deaminase

A variety of bacteria and yeasts were examined for activities of biotin biosynthetic enzymes, including pimelyl-CoA synthetase, 7-keto-8-aminopelargonic acid (KAPA) synthetase, 7,8-diaminopelargonic acid (DAPA) aminotransferase and dethiobiotin (DTB) synthetase. Among the strains tested, only Bacillus sphaericus, a DTB producer, showed significant activities for all four enzymes. The bacterium also exhibited high activity of biotin synthesis from DTB in an intact cell system. Using cell-free extract and intact cells, some properties of DAPA aminotransferase, DTB synthetase and biotin synthesizing reaction were examined.

Based on these results of enzyme activities DTB productivity of B. sphaericus was discussed.     

Adenosine metabolism in wild-type and enzyme-deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5-diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild-type cells, both in intact cells and cell-free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine-hypoxanthine-inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2-deoxycoformycin. Cell-free extracts of the tubercidin-resistant variants, as well as of two adenosine-resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at less than 1% of the rate of extracts of wild-type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40-fold. Similarly, intact adenosine kinase-deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5-10% the rate observed with wild-type cells. Phosphorylation of adenosine and tubercidin in wild-type cells was inhibited by substrate at concentration above 5-10 microM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase-deficient cells increased linearly up to a concentration of 400 microM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild-type cells. Adenosine uptake by these kinase-deficient cells was inhibited by adenine and 5'-deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase-deficient cells via adenine. Indirect evidence implicates 5'-methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.     

Incorporation of the sulfur of L-[S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Studies on Production of Biotin by Microorganisms

During the course of the study on biotin vitamers production by a hydrocarbon-utilizing bacterium, strain 5–2 (Pseudomonas sp.), it was found that crude RNA-alkali-hydrolyzate from yeast increased the accumulaion of biotin vitamers, most of which was determined as desthiobion, and that adenine in the crude RNA-alkali-hydrolyzate was a potent stimulator. Effect of adenine on biotin vitamers accumulation was observed in the medium with either hydrocarbon or glucose as a sole carbon source. The accumulation of total biotin vitamers by some other bacteria was also increased by adenine but that of true biotin was scarcely increased or inhibited by adenine.

The role of adenine on the accumulation of biotin vitamers was investigated with non-proliferating cells of strain 5–2, and it was supposed that adenine would not only inhibit the accumulation of true biotin but, as a result, cause the large accumulation of biotin vitamers which might be intermediates of biotin synthesis. When the medium was supplemented with excess biotin, complete repression occurred even in the presence of adenine.     

Incorporation of the sulfur of L-[35S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Determination of the metabolic origins of the sulfur and 3'-nitrogen atoms in biotin of Escherichia coli by mass spectrometry

Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined. Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin. We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-[sulfane-34S]thiocystine. Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis. Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % [15N]methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N. These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid.     

Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis.

Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli.

We constructed the plasmid pTTB151 in which the E. coli bioB gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe2+, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.     

Convergent pathways of gibberellin A1 biosynthesis in Brassica

[²H, ³H]Gibberellin A₄ (GA₄) or [²H, ³H] GA₉ were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [²H]GA₉ application, [²H]GA₅₁, [²H]GA₂₀ and [²H]GA₄ were identified as products by GC-MS, while [²H]GA₃₄ and [²H]GA₁ were formed from [²H]GA₄. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA₁ biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA₄ or GA₂₀, the latter of which can originate from GA₉ or from GA₁₉.     

3 beta,13-dihydroxylated C20 gibberellins from inflorescences of Rumex acetosa L

Using full scan GC-MS a wide range of gibberellins (GAs) was identified in the young inflorescences of the dioecious species Rumex acetosa L., consistent with the ubiquitous early 13-hydroxylation pathway in both male and female plants. In addition, R. acetosa is the first species in which all three 3beta,13-dihydroxylated C(20)-GAs-GA(18), GA(38) and GA(23)-have been identified in the same organism, suggesting an early 3beta,13-dihydroxylation biosynthesis pathway in this species. Authentic GA(18), GA(38) and GA(23) were synthesized and their effects and that of GA(1), a GA common to both pathways, on the time to inflorescence emergence was investigated. GA(1) accelerated the emergence of inflorescences in both male and female plants. In addition some evidence for biological activity per se of the C(20)-GA(38) was obtained.     

Adenosine suppresses alpha(4)beta(7) integrin-mediated adhesion of T lymphocytes to colon adenocarcinoma cells

The interaction of T lymphocytes with tumor cells, a key step in the antitumor immune response, is suppressed by adenosine, a nucleoside produced at increased levels within the hypoxic tumor environment. We have explored the mechanism by which adenosine interferes with the lymphocyte:tumor cell interaction. The adhesion of anti-CD3-stimulated T cells to syngeneic MCA-38 mouse colon adenocarcinoma cells did not involve LFA-1 (alpha(L)beta(2)) or VLA-5 (alpha(5)beta(1)). However, antibodies against either lymphocyte alpha(4) or beta(7) (but not beta(1)) integrin subunits, or against VCAM-1 on the tumor cells, significantly suppressed adhesion, showing that the recognition of MCA-38 cells by T cells is strongly dependent upon the association of alpha(4)beta(7) on the effector cells with VCAM-1 on the tumor targets. This association is modulated by adenosine: The ability of adenosine to suppress T cell adhesion to MCA-38 cells was lost if alpha(4)beta(7) was functionally blocked with anti-alpha(4) antibodies (i) prior to or (ii) during the adhesion assay or if (iii) alpha(+)(4) cells were depleted from the T lymphocyte population. The binding of T cells to fibronectin through alpha(4)beta(1) was not suppressed by adenosine. We conclude that adenosine partially inhibits the interaction of T lymphocytes with tumor cells by blocking the function of integrin alpha(4)beta(7).     

Biosynthesis of biotin from dethiobiotin by the biotin auxotroph Lactobacillus plantarum.

Lactobacillus plantarum requires biotin for growth. We show that in the presence of high levels of the biotin biosynthetic precursor, dethiobiotin, L. plantarum synthesizes biotin and grows in medium with dethiobiotin but without biotin. Lactobacillus casei also grew under similar conditions.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Assay for biotin in the presence of dethiobiotin with Lactobacillus plantarum

At concentrations greater than approximately 0.5 microM, dethiobiotin can cause the bioassay for biotin, which employs Lactobacillus plantarum, to over value the actual biotin level. This can be as much as 30-fold at 10 microM DL-dethiobiotin and 5 pM biotin. Dethiobiotin does this by exerting a sparing effect on the biotin response by the assay organism. We demonstrate one way to determine the actual biotin concentration in the presence of interfering levels of dethiobiotin.