Incorporation of Angustmycin C,a Nucleosidic Antibiotic,into Ribonucleic Acid in Escherichia coli

A nucleosidic antibiotic angustmycin C, which is an analogue of adenosine and which has an unusual hexose, psicofuranose, instead of ribose, was confirmed to be incorporated into RNA in Escherichia coli. After incubation of cells with tritiated angustmycin C, small but significant radioactivity was found in RNA fraction. When the RNA was hydrolyzed by an enzyme mixture of snake venom, pancreatic ribonuclease I and alkaline phosphatase, intact angustmycin C was recovered which indicated incorporation of the antibiotic into the polyribonucleotide chain of RNA.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

3′, 5′-Cyclic Adenosine Monophosphate Dependent Xylose Lethal Phenomenon in Escherichia coli

The growth of Escherichia coli W2252 was found to be inhibited when xylose and cAMP coexisted in the medium such as peptone or nutrient broth. Among other sugars, only arabinose imposed weaker effect. cAMP could not be replaced by adenine, adenosine, 5′-AMP, 3′-AMP and other 3′,5′-cyclic nucleoside monophosphates. Dose response was observed with reference to either xylose or cAMP. In the presence of both 1% xylose and 10 mm cAMP in peptone broth, 90% of logarithmic phase cells of E. coli W2252 were killed within 6 hr at 37°C. We call this phenomenon as cAMP dependent xylose lethal. This phenomenon was also observed with many substrains of E. coli K–12, E. coli C, Aerobacter aerogenes and Salmonella typhimurium, but not with their xylose negative mutants.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes

Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51?g/L) and UDP-GlcNTFA (46.54?g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.     

Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

A convenient lactic dehydrogenase-coupled assay for determining pyridoxal 5′-phosphate in plasma

An assay for determining the concentration of pyridoxal 5′-phosphate in plasma from 0.4 ml whole blood is reported. The assay consists of incubating deproteinized plasma with d-serine apodehydratase from Escherichia coli in 0.5 mN-2-hydroxyethyl-piperazine-N′-3-propanesulfonic acid, pH 7.8, at 37°C for 15 min, and then determining the d-serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25°C) was used to measure the rate of enzymically catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal 5′-phosphate in the plasma sample was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5′-phosphate present in the assay.     

The synthesis of choline and ethanolamine phosphoglycerides in neuronal and glial cells of rabbit in vitro

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine-5′-phosphate choline (cytidine-5′-phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell-enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine-5′-phosphate choline and cytidine-5′-phosphate ethanolamine, respectively. Several kinetic properties of the choline-phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor cytidine-5′-phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine-dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.     

Production of Guanosine by Psicofuranine and Decoyinine Resistant Mutants of Bacillus subtilis

Growth of Bacillus subtilis AG169 that produced large amounts of xanthosine and guanosine was inhibited by psicofuranine. When AG169 was mutated to resistance against psicofuranine, a mutant, GP–1, which yielded more guanosine was obtained. Psicofuranine did not inhibit growth of GP–1 any more. The guanosine 5′-monophosphate (GMP) synthetase activities were then assayed. In GP–1, the specific activity decreased about half, the complete loss of repression by GMP was found, and the inhibition by GMP was slightly loosed, when compared with those of AG169.

Furthermore, as growth of GP–1 was strongly inhibited by decoyinine, decoyinine resistant mutants were derived from GP–1. Of these mutants, two strains, MG–1 and MG–4, were resistant to decoyinine completely and showed the exclusive accumulation of guanosine in high yields, i.e. 16.0 and 15.5 g of guanosine per liter with weight yields of 20.0 and 19.4% of consumed sugar, respectively. GMP synthetase activity of MG–1 increased remarkably in comparison with that of GP–1 or AG169, and the inhibitions by GMP, psicofuranine and decoyinine were completely released in MG–1. Namely, the psicofuranine and decoyinine resistances seemed to cause mainly variations of GMP synthetase, and as results, the conversion of xanthosine 5′-monophosphate (XMP) to GMP proceeded more smoothly, and a larger amount of guanosine was accumulated.     

Theoretical drug design: 6-azauridine-5'-phosphate--its X-ray crystal structure, potential energy maps, and mechanism of inhibition of orotidine-5'-phosphate decarboxylase.

The cytostatic drug 6-azauridine is converted in vivo to 6-azauridine-5′-phosphate (z⁶Urd-5′-P), which blocks the enzyme orotidine-5′-phosphate decarboxylase (Ord-5′-Pdecase) and therefore inhibits the de novo production of uridine-5′-phosphate (Urd-5′-P). In order to relate the structure and function of z⁶Urd-5′-P, it was crystallized as trihydrate, space group P2₁2₁2₁ with a = 20.615 Å, b = 6.265 Å, c = 11.881 Å, and the structure established by Patterson methods. Atomic parameters were refined by full-matrix least-squares methods to R = 0.066 using 1638 counter measured x-ray data. The ribose of z⁶Urd-5′-P is in a twisted C(2′)-exo, C(3′)endo conformation, the heterocycle is in extreme anti position with angle N(6)-N(1)-C(1′)-O(4′) at 86.3°, and the orientation about the C(4′)-C(5′) bond is gauche, trans in contrast to gauche, gauche found for all the other 5′-ribonucleotides. Conformational energy calculations show that z⁶Urd-5′-P may adopt an extreme anti conformation not allowed to Urd-5′-P, and they also predict the same unusual trans, gauche conformation about the C(4′)-C(5′) bond in orotidine-5′-phosphate (Ord-5′-P) and in z⁶Urd-5′-P, which renders the distances O(2)…O(5′) in z⁶Urd-5′-P and O(7)…O(5′) in Ord-5′-P comparable. On this basis the function of z⁶Urd-5′-P as an Ord-5′-Pdecase inhibitor can be explained as being due to its structural similarity with the substrate Ord-5′-P and further clarifies the inhibitory action of 5′-nucleotides bearing the heterocycles oxipurinol, xanthine, or allopurinol [J. A. Fyfe, R. L. Miller, and T. A. Krenitsky, J. Biol. Chem. 248 , 3801 (1973)]. With this in mind, new inhibitors for Ord-5′-Pdecase may be designed.     

Microbial Synthesis of Coenzyme A Intermediates

Greater production of pantothenic acid 4′-phosphate and pantetheine 4′-phosphate by a microorganism were described. The incubation of pantothenic acid and adenosine 5′-triphosphate with resting cells of Brevibacterium ammoniagenes IFO 12071 gave pantothenic acid-4′-phosphate in a high yield. Cultivation of the organism with pantothenic acid and 5′adenylic acid also gave pantothenic acid 4′-phosphate in a high yield. In a similar fashion pantetheine 4′-phosphate was readily obtained in a good yield. The products were identified chemically and enzymatically.     

Biosynthesis of polyribonucleotide phosphorylase and polyribonucleotides by E. Coli

The biochemical functions of Polyribonucleotide phosphorylase (PNP-ase; EC 2.7.7.8) has been studied The present work was aimed at studying the interrelation between PNP-ase biosynthesis and RNA content of E. coli cells under various conditions of bacterial growth, obtaining the biomass of E. coli with high PNP-ase activity as well as to study the possibility of secondary cultivation of E. coli cells for obtaining polyribonucleotides from nucleoside-5′-diphosphates The spcific PNP-ase activity increases at secondary cultivation, when the medium contains ribonucleoside-5′-diphosphates. Besides one may observe an extracellular polycondensation of nucleoside-5′-diphosphates It may be presumed that the PNP-ase is a multifunctional enzyme whose biological role may be confined to regulating the energetic processes connected with the metabolism of glucose, nucleosidephosphates and orthophosphate in the cells of E. coli.     

The Sites of Action of Allopurinol in Crithidia fasciculata*

Reversal of the growth inhibition of Crithidia fasciculata by allopurinol requires both a purine and a pyrimidine. Hypoxanthine is the most effective purine in the reversal. Cell-free extracts were prepared which were capable of the decarboxylation of orotidine 5′-phosphate. Other enzyme preparations carried out the phosphoribosylation of allopurinol. By the use of [4-¹⁴C] orotidine 5′-phosphate (enzymatically prepared), it was shown that allopurinol ribotide (enzymatically prepared), but not the free base, inhibits orotidine 5′-phosphate decarboxylase.     

Aminoacyl-tRNA Analogues; Synthesis,Purification and Properties of 3′-Anthraniloyl Oligoribonucleotides

Abstract

Reaction of isatoic anhydride with adenosine, adenosine 5′-phosphate, oligoribonucleotides or with the E. coli tRNA^Val led to attachment of an anthraniloyl residue at 2′-or 3′-OH groups of 3′-terminal ribose residue. No protection of the S'-hydroxyl group or internal 2′-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Partial Purification and Some Properties of Aggregation Factor from Pronase-Susceptible Floe Forming Flavobacterium Strain B

Globomycin inhibited the incorporation of [¹⁴C]diaminopimeric acid (Dap) into the cold 5% TCA insoluble fraction of Escherichia coli H2143 at higher concentrations than the minimum inhibitory concentration (MIC).

One-sixth or -seventh molecules of the lipoprotein were found per one molecule of N-acetyl glucosamine (GlucNAc) or Dap in globomycin-treated cells as compared with one-twelfth or -thirteenth molecules in normal cells. Among globomycin-resistant cells isolated, one-tenth were lipoprotein-less mutants and they showed a slightly swollen form and leaked RNase into the medium. It was interesting that spheroplast formation of the mutants in the presence of the antibiotic was not observed even at a high concentration.