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Epimerization of Thymidine Diphosphate Glucose in Bacterial Extracts Regina Tinelli,1 A

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Epimerization of Thymidine Diphosphate Glucose in Bacterial Extracts Regina Tinelli,1 A EPIMERIZATION OF THYMIDINE DIPHOSPHATE GLUCOSE IN BACTERIAL EXTRACTS REGINA TINELLI,1 A. M. MICHELSON,2 AND JACK L. STROMINGER Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri Received for publication 27 March 1963 ABSTRACT TDP-D-glucose was also converted to TDP-D- TINELLI, REGINA (Washington University galactose by several of these extracts: School of Medicine, St. Louis, Mo.), A. M. TDP-D-glucose > TDP-D-galactose (2) MICHELSON, AND JACK L. STROMINGER. Epimeri- Pasteurella zation of thymidine diphosphate glucose in pseudotuberculosis type IV was chosen for more bacterial extracts. J. Bacteriol. 86:246-251. detailed investigation, because 1963.-Extracts of Pasteurella this organism was not known to contain rhamnose pseudotuberculosis (Davies, 1960) and novel of type IV and several other bacteria catalyzed the pathways TDP- sugar metabolism reversible interconversion of thymidine diphos- might, therefore, occur. This of a phate (TDP)-D-glucose and TDP-D-galactose. paper, which preliminary account has been Moreover, extracts of P. pseudotuberculosis published (Tinelli et al., 1962), is a detailed account of these experiments. Epimerization of cata1yzzd the synthesis of TDP-D-glucose from TDP-D-glucose has also been reported to occur thymidine triphosphate and a-D-glucose-1-phos- in phate. The limitations of these observations and plants (Neufeld, 1962) and in galactose- adapted S. faecalis (Pazur, Kleppe, and Cepure, their possible significance are discussed. 1962). Thymidine diphosphate (TDP) sugar com- MATERIALS AND METHODS pounds were first isolated in 1959 (Okazaki, Bacteria. All bacteria were harvested during 1959, 1960; Strominger and Scott, 1959), and, logarithmic growth. The cells obtained from 1 since then, a variety of other compounds of this liter (1.5 to 2.5 g) in 25 ml of 0.02 M tris(hydroxy- type have been isolated or synthesized enzy- methyl)aminomethane (tris) buffer (pH 7.8) matically, or both. TDP-D-glucose is synthesized were treated for 20 min in a Raytheon 10-ke in bacteria from thymidine triphosphate (TTP) sonic oscillator. This sonic extract was centrifuged and a-D-glucose-l-phosphate (Pazur and Shuey, at 30,000 X g for 10 min. The supernatant solu- 1961; Kornfeld and Glaser, 1961): tion was used as the enzyme. In some experi- ments where glucose formation was measured, TTP + glucose-l-phosphate endogenous substrates were removed by passing 1 TDP-glucose + pyrophosphate ml of the extract over a 10-ml column of Sephadex It can be reduced to G-25 in 0.02 M tris buffer (pH 7.5). TDP-L-rhamnose (Pazur Nucleotides. TDP-D-galactose and Shuey, 1961; Glaser and Kornfeld, 1961) was prepared in a synthetically by reaction of a-D-galactose-l- complex reaction sequence in which TDP-4- phosphate with diphenyl keto-6-deoxy-D-glucose (Okazaki et aL., 1962) TDP, and subsequent is an purification, as previously described for prepara- intermediate. This transformation occurs tion of synthetic in Escherichia coli, Pseduomonas aeruginosa, TDP-D-glucose (Okazaki et al., Streptococcus faecalis, and other 1962). Nucleotides were adsorbed onto charcoal bacteria. During and handled for various analyses as described the course of investigation of this reaction se- previously quence in various it (Okazaki et al., 1962). bacteria, was observed that Paper chromatography was carried out in 1 Present address: Institut Pasteur, Paris, solvent A: ethyl acetate-pyridine-water (36:10: France. 11.5); solvent B: isobutyric acid-0.5 N NH40H 2 Present address: Institut de Biologie Physico- (5:3); or solvent C: ethanol-i M ammonium Chimique, Paris, France. acetate, pH 7 (7.5:3). Nucleotides were visualized 246 VOL. 86, 1963 EPIMERIZATION OF THYMIDINE DIPHOSPHATE GLUCOSE 247 under ultraviolet light. Sugars were detected with of the nucleotides from the incubation mixture) alkaline silver nitrate (Anet and Reynolds, 1954). with the mobility of galactose in solvent A Analytical methods. Hexose and 6-deoxyhexose (mobility relative to glucose = 0.86). These or- were measured by the 3-min cysteine-H2SO4 ganisms included both smooth and rough strains reaction (Dische and Shettles, 1951). TDP-D- of E. coli 08, Salmonella berlin, P. pseudotubercu- glucose was measured using the enzyme from losis type IV, and P. pseudotuberculosis type E. coli B, treated with Sephadex G-25, which V. Extracts of these organisms could not catalyze converts this nucleotide to TDP-4-keto-6-deoxy- the formation of TDP-L-rhamnose. (An extract of D-glucose (Okazaki et al., 1962). The incubation the rough strain of S. berlin catalyzed formation of reagent (60,uliters) contained tris buffer (40 mM), TDP-L-rhamnose at a slow rate and is an excep- ethylenediamiiinetetraacetate (EDTA; 1 mM), tion to this statement.) No galactose was formed cysteine (3 mM), MgCl2 (10 mM), and 0 to 50 by extracts of those organisms in which TDP-L- m,umoles of TDP-D-glucose. Sephadex-treated rhamnose or TDP-4-keto-6-deoxy-D-glucose was E. coli B extract (40 Aliters) was added, and the formed; these strains included E. coli B, E. coli mixture was incubated at 37 C for 1 to 2 hr. Y-10, Alcaligenes faecalis LB, and smooth and Then 0.1 ml of 0.15 N NaOH was added, and, rough strains of E. coli 018 and S. weslaco. after 20 min at room temperature, the absorbancy Since reactions of TDP-sugar compounds had at 320 m,u due to TDP-4-keto-6-deoxy-D-glucose not previously been observed in organisms which was measured. This sensitive measurement of could not form TDP-L-rhamnose or its precursors, TDP-D-glucose was linear in the range indicated P. pseudotuberculosis type IV was chosen for (Fig. 1). further study. These and other experiments indi- Glucose was measured with D-glucose oxidase cated that the smooth and rough strains of P. (Huggett and Nixon, 1957), or by phosphoryla- pseudotuberculosis formed galactose in this reac- tion and oxidation to 6-phosphogluconic acid us- ing a fluorometric procedure (Lowry and Passon- 0.550 neau, in press). The sample (2 to 20 m,umoles of glucose) was added to 1 ml of a solution in a flou- I rometer tube containing tris buffer, pH 8.0 (100 0 a mM), nicotinamide adenine dinucleotide phos- z phate (NADP; 0.05 mM), MgCl2 (5 mm), aden- ZI osine triphosphate (ATP; 0.3 mM), bovine serum albumin (0.01%), hexokinase (Boehringer, 2 0 ,lAiters of 5 mg/ml), and glucose-6-phosphate z 0. dehydrogenase (Boehringer, 5 ,uliters of 0.8 E mg/ml). 0 Appearance of reduced NADP flourescence CM was measured as described by Lowry, Roberts, 0.2! and Kapphahn (1957), and the reaction was com- 0 plete in 3 to 4 min. D-Galactose was measured z with D-galactose dehydrogenase of Pseudomonas saccharophila, as employed previously (Kruger 0 0.150[ et al., 1962). mcl: RESULTS Formation of galactose from TDP-D-glucose in 0.< various bacteria. During studies of TDP-L- rhamnose biosynthesis in various bacteria (Okazaki et al., 1962; Okazaki, Strominger, and 0 20 40 60 Okazaki, 1963), it was observed by Mrs. Tuneko m,umoles of TDP- GLUCOSE Okazaki that extracts of several of the organisms FIG. 1. Measurement of TDP-D-glucose by con- employed catalyzed the formation from TDP-D- version to TDP -4- keto -6- deoxy - D - glucose with glucose of a sugar (obtained by acid hydrolysis enzyme from Escherichia coli B. 248 TINELLI, MICHELSON, AND STROMINGER J. BACTERIOL. tion at nearly the same rates. Subsequent experi- 0.73). A sample was hydrolyzed in 0.1 N HCl, ments were, therefore, carried out with the and then HCl was removed in vacuo. Paper rough strain of this organism. chromatography in solvent A revealed the Isolation and identification of TDP-hexoses presence of two sugars with the mobilities of after incubation of TDP-D-glucose with extract of P. glucose and galactose. These sugars were further pseudotuberculosis type IV. Two-dimensional identified by specific enzymatic reactions. In a paper chromatography, as described by Okazaki solution of the nucleotide which contained 1.8 et al. (1963), was used to isolate the products ,umoles of thymidine per ml (estimated from of this reaction. TDP-D-glucose (3,imoles) was its absorbancy at 267 min), 1.3 ,umoles/ml of incubated in 0.8 ml of solution containing tris D-glucose, measured with D-glucose oxidase, buffer (pH 7.7, 20 mM) disodium EDTA (0.5 and 0.3 ,umole/ml of D-galactose, measured with mM), cysteine (1 mM), MgCl2 (5 mM), and 0.35 D-galactose dehydrogenase purified from D- ml of sonic extract of P. pseudotuberculosis type galactose-adapted P. saccharophila, were found. IV (10 mg of protein/ml). After 3 hr at 37 C, Formation of TDP-glucose by extract of P. the reaction mixture was placed in a boiling- pseudotuberculosis type IV. Glucose-i-phosphate water bath for 1 min. Coagulated protein was (2 ,umoles) and 0.5 ,umole of TTP were incubated removed by centrifugation, and the pH was in 0.2 ml of solution containing tris buffer (20 adjusted to 5. Then the nucleotides were adsorbed mM), disodium EDTA (0.5 mM), cysteine (1.5 into charcoal and eluted with ammoniacal mM), MgCl2 (2.5 mM), NaF (10 mM), MnCl2 ethanol. After two-dimensional chromatography (2.5 mM), and 0.05 ml of sonic extract of P. in solvents B and C, a single ultraviolet-absorbing pseudotuberculosis type IV. After incubation for 3 compound was observed in the position of TDP- hr at 37 C, nucleotides were recovered from the hexoses. This compound was eluted from paper. incubation mixture and subjected to paper Its spectrum was identical to that of a thymidine chromatography as described above.
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