Isolation and Characterization of a Cyclic Phosphate of Neopterin and Other 6-Alkylpterin Compounds Enzymatically Synthesized from GTP by Cell-free Extracts of Serratia indica

A cell-free system for the biosynthesis of l-threo-neopterin, a growth factor for protozoan, Crithidia fasciculata from guanosine-5′-triphosphate (GTP) was obtained from extracts of Serratia indica IFO 3759. This preparation catalyzed the production of a specific pteridine from GTP, which was isolated and characterized as a cyclic phosphate of neopterin (cNP). Among the other products, l-threo-neopterin, as the Crithidia factor, 6-hydroxymethylpterin, and erythro-neopterin were tentatively identified. Requirements for the synthesis of these products include GTP, Mg²⁺, and disodium phosphate. Fluorescence formation was inhibited by purine nucleotides.

When a disodium phosphate was included in the reaction system, cNP and erythro-neopterin were effectively synthesized from GTP. On the other hand, when the phosphate was omitted 6-hydroxymethylpterin was formed.

The possible biosynthetic process of l-threo-neopterin was discussed.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

Isolation and Identification of Crithidia Factors from Bacillus cereus IFO 3131

Bacillus cereus IFO 3131 produces the largest amount of Crithidia factors of 27 bacterial species tested (J. Bacteriol., 104, 197, 1970). The factors in the culture fluid and cell were isolated with a Florisil column and reversed-phase high-performance liquid chromatography (HPLC). They were identified by HPLC, fluorescence and ultraviolet spectra, thin-layer chromatography and bioassay. The major factor in the cell was d-erythro-neopterin and that in fluid was 6-hydroxymethylpterin and pterin.     

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.     

The Mutational Effect of Ile58 at Subsite C in Hen Egg-White Lysozyme on Substrate Binding,Enzymatic Activity,and Protein Stability

Partial acid hydrolysis of Saccharomyces cerevisiae mannan gave 2-O-α-d-Manp-d-Man (1), 3-O-α-d-Manp-d-Man (2), 6-O-α-d-Manp-d-Man (3), O-α-d Manp-(1→2)O-α-d-Manp-(1→2)-d-Man (4), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-d-Man (5), O-α-d Manp-(1→6)-6-O-α-d-Manp-(1→6)-d-Man (6), O-α-d Manp-(1→2)-O-α-d-Manp-(1→2)-6-O-α-d-Manp-(1→6)-d-Man (7), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-O-α-d-Manp-(1→6)-d-Man (8), and O-α-d-Manp-(1→6)-O-[α-d-Manp-(1→2)]-O-α-d-Manp-(1→6)-d-Man (9).     

Preparation of [1-13C]D-Glucose 6-Phosphate from [13C]Methanol and D-Ribose 5-Phosphate with Methylotrophic Enzymes

[¹³C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-¹³C]D-fructose 6-phosphate produced in this reaction was converted to [1-¹³C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [¹³C]methanol was used as the C₁-donor, the yield of [1-¹³C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-¹³C]D-glucose 6-phosphate and 16.4g/liter [1-¹³C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H₂O₂ and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

A Novel Reaction of Sugars with Anion Radical of Carbon Dioxide Produced from Formate

Radiolysis of some monosaccharides (fructose, glucose and ribose) in air-free condition was markedly enhanced by the addition of formate at concentrations above 20 mm, while it was inhibited at concentrations below 20 mm. The following compounds were detected in the irradiated sugar solutions containing excess formate (100mm): 1-Deoxy-d-arabinohexulose (1, G=4.4) and 1,3- dideoxy-d-erythrohexulose (2, G= 1.3) from fructose; 2-deoxy-d-ribose (3, G=2.3) and 2-deoxyribitol (4, G =0.6) from ribose; and 2-deoxy-d-glucose (5, G=0.5) and 2-deoxy-d-glucitol (6, G=0.4) from glucose. A mechanism for radiolytic formation of the products was proposed, based on interaction of - formed from formate with sugars.     

Characterization of an Extracellular Polysaccharide Elaborated by TX-1, a New Strain of Beijerinckia indica

An extracellular polysaccharide elaborated by a new species of Beijerinckia indica, named TX-1, was composed of D-glucose, L-fucose, D-glycero-D-manno-heptose, and D-glucuronic acid in a molar ratio of 5.0:1.0:2.0:0.9, in addition to 16.2% of the acetyl group. Among the polysaccharides of the Beijerinckia species, the present polysaccharide might be the first acidic type having an L-fucose residue. A methylation analysis, Smith degradation study and fragmentation analysis show that this polysaccharide consisted of non-reducing terminal D-glucose, O-4 substituted D-glucose, O-2 substituted D-glycero-D-manno-heptose, O-4 substituted D-glucuronic acid, O-3 and O-4 substituted D-glucose, and O-3 substituted L-fucose residues. A D-glucuronic acid residue was linked to the O-3 position of the L-fucose residue by an α-glycosidic linkage. Most of the D-glucose residues in the backbone chain were substituted at the O-3 position, with the side chain having non-reducing terminal D-glucose residues. It is suggested by the reaction with Con A that the anomeric configuration of the terminal D-glucose residues was β.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

Synthesis of Certain Disaccharides Containing a α-or β- Rhamnopyranosidic Group and the Substrate Specificity of α-l-Rhamnosidase from Aspergillus niger

To investigate the substrate specificity of α-l-rhamnosidase from Aspergillus niger, the following seven substrates were synthesized: methyl 3-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (1), methyl 3-O-α-l-rhamnopyranosyl-α-l-xylopyranoside (2), methyl 3-0-α-l-rhamnopyranosyl-α-l-rhamnopyranoside (3), methyl 4-0-α-l-rhamnopyranosyl-α-d-galactopyranoside (4), methyl 4-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (5), methyl 4-0-α-l-rhamnopyra-nosyl-α-d-xylopyranoside (6), and 6-0-β-l-rhamnopyranosyl-d-mannopyranose (7). Compounds 1~6 were well-hydrolyzed by the crude enzyme, but 7 was unaffected.     

Coenzyme Q10 in the Respiratory Chain Linked to Fructose Dehydrogenase of Gluconobacter cerinus

A glucomannan isolated from konjac flour was hydrolyzed with commercially available crude and purified cellulases. The following oligosaccharides were isolated from the hydrolyzate and identified: (a) 4-O-β-d-mannopyranosyl-d-monnose (b) 4-O-β-d-mannopyranosyl-d-glucose (c) O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose (d) O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-glucose (e) O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose (f) O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-glucose (g) O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-glucose (h) 4-O-β-d-glucopyranosyl-d-glucose(cellobiose) (i) 4-O-β-d-glucopyranosyl-d-mannose (epicellobiose) (j) O-β-d-glucopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose. Of these saccharides, (h), (i) and (j) were isolated from the hydrolyzate by purified cellulase, while (g) was isolated from the hydrolyzate by crude cellulase. The others were all present in the hydrolyzates both by crude and by purified cellulases.     

Biotransformation of Cinnamic Acid,p-Coumaric Acid,Caffeic Acid,and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana

Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid β-D-glucopyranosyl ester, p-coumaric acid, and 4-O-β-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-β-D-glucopyranosylcoumaric acid, p-coumaric acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcoumaric acid β-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-β-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-β-D-glucopyranosylcaffeic acid, 3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosylcaffeic acid, a new compound, 3-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcaffeic acid, 4-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, ferulic acid, and 4-O-β-D-glucopyranosylferulic acid. 4-O-β-D-Glucopyranosylferulic acid, ferulic acid β-D-glucopyranosyl ester, and 4-O-β-D-glucopyranosylferulic acid β-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Synthesis and Biological Activity of the Lipophilic Derivatives of N- Acetyl-1-thiomuramoyl-l-alanyl-d-isoglutamine

A variety of the lipophilic derivatives at C-1 and C-6 in N-[2-O-(2-acetamido-2,3-dideoxy-1-thio-β-d-glucopyranose-B-yl)-d-lactoy]-l-alanyl-(N¹-fatty acyl)-d-isoglutamine methyl esters were synthesized from 2N-acetyl-1-S-acetyl-4,6-O-isopropylidene-1-thiomuramoyl-l-alanyl-d-isogluta-mine methyl ester. Their immunoadjuvant activity in guinea-pigs, and the protective effect in mice infected with Escherichia coli (E-77156) were examined.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Ng-Methylated Arginines in Broad Bean Seed

l-N^g-Methylarginine, l-N^g,N^g-dimethylarginine and ethanolamine were isolated from basic amino acids fraction of broad bean (Vicia faba L.) seed. The presence of N^g,N′^g-dimethylarginine was also suggested.     

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various sugars and sugar alcohols. d-Mannose, d-glucosamine, N-acetyl- d-glucosamine, d-xylose, d- allose, isomaltose, and cellobiose were efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymic methods that this enzyme could transfer glycosyl residues only to the C4-hydroxyl groups of d-mannose, iY-acetyl- d-glucosamine, d-allose, and d-xylose, producing oligosaccharides terminated by 4–0-α-d-glucopyranosyl-d-mannose, 4–0-α-d-glucopyranosyl-yV-acetyl-d-glucosamine, 4-O-α-d-glucopyranosyl-d-allose, and 4–0-α-d-gluco- pyranosyl-d-xylose at the reducing ends, respectively.