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. 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 experiments 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 reaction and oxidation to 6-phosphogluconic acid using 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. A small nucleotide (Amax in acid = 267 MA; A280/A260 = amount of an ultraviolet-absorbing spot was found in the location of TDP-hexoses. This compound was eluted. It had the spectrum of a 1.0 thymidine nucleotide (Fig. 2). It was identified as TDP-D-glucose by conversion to TDP-4- keto-6-deoxy-D-glucose and, after acid hydrolysis, by oxidation with D-glucose oxidase. The sample 0.8F analyzed contained 0.074 ,umole/ml of TDP-D- glucose by the former method, 0.083 MAmole/ml of D-glucose by the latter method, and 0.087 - 0.72 ,umole/ml of thymidine nucleotide. 0.6F A260 Conversion of TDP-D-galactose to TDP-D- z glucose. Since measurement of formation of TDP-D-galactose from TDP-D-glucose was lim- 0In U, ited by the availability of D-galactose dehydro- In0 0.4F genase, a convenient assay for the enzyme was developed based on the conversion of synthetic TDP-D-galactose to TDP-D-glucose. These experiments also illustrated the reversibility of the reaction. The TDP-D-glucose formed was 0.2F readily measured by acid hydrolysis, followed by fluorometric measurement of D-glucose in the presence of ATP, hexokinase, NADP, and D-glucose-6-phosphate dehydrogenase. 240 260 280 300 320 For the assay, 2 jAliters of TDP-D-galactose WAVELENGTH, m/u (10 ,umoles/ml) were added to 25 ,uliters of FIG. 2. Absorption spectrum in 0.01 N HCJ of buffer reagent [80 mm tris buffer (pH 7.7), 2 mM enzymatically synthesized TDP-D-glucose. EDTA, 10 mM cysteine, and 20 mM MgCl2]. Sonic VOL. 86, 1963 EPIMERIZATION OF THYMIDINE DIPHOSPHATE GLUCOSE 249 extract (25 ,uliters) was then added. After incubation at 37 C, 0.1 ml of 0.15 N HCl was added. The .500 q a tubes were placed in a boiling-water bath for 15 w -Q min, and then centrifuged. The supernatant was * 0 removed and neutralized by the addition of Li. owwn 10 lliters of 1.5 N NaOH. Glucose was then meas- .0a ured fluorometrically. Under these conditions, 300 a wO kz formation of glucose was proportional to time in U)I o -< the early stages of the incubation (Fig. 3) and 443@3 '-C (AJ to amount of enzyme added. At equilibrium, n o ro 0 about one-half of the TDP-D-galactose was con- E 1.100 c X verted to TDP-D-glucose. 0Co The activity of the sonic extract measured in E (S this way corresponded to 50 m,umoles per mg of protein per hr (750 miumoles per ml per hr). TIME, HOURS With uridine diphosphate (UDP)-D-galactose as FIG. 4. Simultaneous mteasurement of TDP-D- substrate, using the same assay the activity glucose and TDP-4-keto-6-deoxy-D-glucose forma- was 5 to 10 times higher. About a tenfold purification in extracts of Escherichia coli B incubated with tion was obtained by two precipitations with TDP- D-galactose. ammonium sulfate between 28 and 45% and then between 32 and 44%. The UDP-D-glucose from TDP-D-glucose (Tinelli et al., 1962). This epimerase activity was unaltered by this purifica- result, however, is due to the presence of the tion. enzyme which irreversibly forms TDP-4-keto-6- The epimerization of TDP-D-galactose cata- deoxy-D-glucose from TDP-D-glucose. Since lyzed by the original extract or by the purified TDP-D-galactose formation is reversible, in long enzyme was not stimulated by 10-4 M nico- incubations TDP-D-glucose is converted quantita- tinamide adenine dinucleotide (NAD), nor could tively to TDP-4-keto-6-deoxy-D-glucose. This a NAD requirement be demonstrated after phenomenon can readily be demonstrated using passage of the enzyme over a column of Sephadex TDP-D-galactose as the substrate. For this ex- G-25. periment, TDP-D-glucose formation was meas- Epimerization of TDP-D-galactose in extracts of ured, after acid hydrolysis, in a sample of the E. coli B. It has been earlier noted, employing incubation mixture with hexokinase and glu- paper chromatography of a hydro]ysate of the cose-6-phosphate dehydrogenase. TDP-4-keto-6- nucleotides, that several strains of E. coli, which deoxy-D-glucose was measured in a second contain a UDP-D-glucose epimerase, did not sample by addition of 0.1 N NaOH followed by form any detectable amount of TDP-D-galactose determination of the absorbancy at 320 m,u. TDP-D-glucose was first formed in the incubation and then disappeared as the amount of

E - +TDP- galactose TDP-4-keto-6-deoxy-D-glucose steadily increased 0 ( 18.3 mej moles ) (Fig. 4). o_ 6 DIscussIoN U) '4 0 24 The physiological significance of the epimerization of TDP-D-glucose is not clear, although this E 2 -TDP- galactose activity is present in extracts of a variety of E bacteria. It is possible that this reaction can be 0 1 2 catalyzed by the same protein which catalyzes TIME, HOURS the epimerization of UDP-D-glucose (Maxwell, Even if this were the a FIG. 3. Formnation of TDP-D-glucose from TDP- 1961). case, however, D-galactose. After incubation with TDP-D-galac- physiological role for formation of TDP-D- tose, the nucleotide mixtuires were hydrolyzed in acid galactose would not be excluded, since there are and D-glucose was measured. several examples of different reactions important 250 TINELLI, MICHELSON, AND STROMINGER J. BACTERIOL. to the economy of the organism and catalyzed matic synthesis of thymidine-linked sugars. by the same protein. Similar considerations II. Thymidine diphosphate L-rhamnose. J. apply to the demonstration of a mechanism for Biol. Chem. 235:1795-1799. formation of TDP-D-glucose in P. pseudotuber- HUGGETT, A. ST. G., AND D. A. NIXON. 1957. Use These have also been raised of glucose oxidase, peroxidase, and O-dianisi- culosis. questions dine in determination of blood and urinary with respect to these reactions in plants (Neu- glucose. Lancet 2:368-370. feld, 1962), and to several other reactions of TDP- KORNFELD, S., AND L. GLASER. 1961. The enzy- sugar compounds in bacteria (Kornfeld and matic synthesis of thymidine-linked sugars. Glaser, 1962). In galactose-adapted S. faecalis, I. Thymidine diphosphate glucose. J. Biol. however, the synthesis of an adaptive enzyme, Chem. 236:1791-1794. TDP-galactose pyrophosphorylase, has been KORNFELD, S., AND L. GLASER. 1962. The synthe- demonstrated (Pazur et al., 1962), and it seems sis of thymidine-linked sugars. V. Thymidine clear in this case that the epimerization is re- diphosphate-amino sugars. J. Biol. Chem. quired for formation of TDP-D-glucose from 237:3052-3059. TDP-D-galactose and subsequent utilization of KRUGER, L., 0. LUDERITZ, J. L. STROMINGER, AND 0. WESTPHAL. 1962. Zur Immunchemie this compound for TDP-L-rhamnose biosynthesis der O-Antigene von Enterobacteriaceae. VII. and for incorporation of L-rhamnose into cell- Die Zugehorigkeit von Hexosen und 6-desoxy- wall polysaccharides. hexosen in Salmonella-Lipopolysacchariden Despite these limitations, however, it may be zur D-bzw. L-Reihi. Biochem. Z. 335:548-558. noteworthy that P. pseudotuberculosis type IV LOWRY, 0. H., N. R. ROBERTS, AND J. I. KAP- is the first example of a microorganism which PHAHN. 1957. The fluorometric measurement can catalyze reactions involving the formation of pyridine nucleotides. J. Biol. Chem. 224: and metabolism of TDP-sugar compounds and 1047-1064. which is not known to contain L-rhamnose or its MAXWELL, E. S. 1961. Enzymic epimerizations, precursors. Extracts of this microorganism, p. 443-453. In P. D. Boyer, H. Lardy, and K. Myrback [ed.], The enzymes, vol. 5. Academic moreover, are unable to synthesize TDP-L- Press, Inc., New York. rhamnose or TDP-4-keto-6-deoxy-D-glucose (a NEUFELD, E. F. 1962. Formation and epimeriza- precursor of TDP-L-rhamnose). These observa- tion of TDP-D-galactose catalyzed by plant tions could suggest a wider role for TDP-sugar enzymes. Biochem. Biophys. Res. Commun. compounds in microbial metabolism. 7:461-466. OKAZAKI, R. 1959. Isolation of a new deoxyribosi- ACKNOWLEDGMENTS dic compound, thymidine diphosphate rham- This work has been supported by research nose. Biochem. Biophys. Res. Commun. 1:34- grants from the U.S. Public Health Service 38. (NIAMD A-1158) and the National Science OKAZAKI, R. 1960. Studies of deoxyribonucleic acid synthesis and cell growth in the deoxyribo- Foundation (G-18742). We also wish to thank side-requiring bacteria, Lactobacillus acido- D. A. L. Davies, Microbiological Research philus. III. Idei'tification of thymidine di- Establishment, Porton, Wilts, England, for phosphate rharnnose. Biochim. Biophys. cultures of the strains of P. pseudotuberculosis Acta 44:478-490. used in this study. OKAZAKI, R., T. OKAZAKI, J. L. STROMINGER, AND A. M. MICHELSON. 1962. Thymidine diphos- LITERATURE CITED phate 4-keto-6-deoxy-D- glucose, an inter- ANET, E. F. L. J., AND T. M. REYNOLDS. 1954. mediate in thymidine diphosphate L-rham- Isolation of mucic acid from fruits. Nature nose synthesis in Escherichia coli strains. J. 174:930. Biol. Chem. 237:3014-3026. DAVIES, D. A. L. 1960. Polysaccharides of gram- OKAZAKI, T., J. L. STROMINGER, AND R. OKAZAKI. negative bacteria. Advan. Carbohydrate 1963. Thymidine diphosphate-L-rhamnose me- Chem. 15:271-340. tabolism in smooth and rough strains of DISCHE, Z., AND L. B. SHETTLES. 1951. A new spec- Escherichia coli 018 and Salmonella weslaco. trophotometric test for the detection of J. Bacteriol. 86:120-126. methylpentose. J. Biol. Chem. 192:579-582. PAZUR, J. H., K. KLEPPE, AND A. CEPURE. 1962. GLASER, L., AND S. KORNFELD. 1961. The enzy- The synthesis of thymidine diphosphate VOL. 86,1963 EPIMERIZATION OF THYMIDINE DIPHOSPHATE GLUCOSE 251

hexoses by Streptococcus faecalis grown on of thymidine diphosphosugar compounds from D-galactose. Biochem. Biophys. Res. Commun. Escherichia coli. Biochim. Biophys. Acta 35: 7:157-161. 552-553. PAZUR, J. H., AND E. W. SHUEY. 1961. The enzy- TINELLI, R., T. OKAZAKI, R. OKAZAKI, AND J. L. matic synthesis of thymidine diphosphate STROMINGER. 1962. Enzymatic synthesis of glucose and its conversion to thymidine di- thymidine diphosphate D-galactose from phosphate rhamnose. J. Biol. Chem. 236: thymidine diphosphate D-glucose in bacterial 1780-1785. extracts. Intern. Congr. Microbiol. 8th Mon- STROMINGER, J. L., AND S. S. SCOTT. 1959. Isolation treal, p. A 1.10.