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Biochemical Conversion of Uridine Diphosphate Glucose to Uridine Diphosphate 3-Ketoglucose by Washing Cells of Agrobacterium Tumefaciens

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Biochemical Conversion of Uridine Diphosphate Glucose to Uridine Diphosphate 3-Ketoglucose by Washing Cells of Agrobacterium Tumefaciens Short Communication [Agr. Biol. Chem., Vol. 34, No. 2, p. 321324, 1970] Biochemical Conversion of Uridine Diphosphate Glucose to Uridine Diphosphate 3-Ketoglucose by Washing Cells of Agrobacterium tumefaciens Sugar phosphate had been generally assumed phosphate permease+) which is a mutant to be impermeable through surface layer of derived from a parent strain Agrobacterium bacterial cells. Recently, however, the ex tumefaciens IAM 1525 (Strs, glucose+, sucrose+, istence of inducible active transport system glucose 1-phosphate+) was used in this experi for L-ƒ¿-glycerophosphate and glucose 6- ment. Standard culture medium had the phosphate was demonstrated in Escherichia following composition: (NH4)2 SO4 100 mg, coli.1•`4) In our previous papers,5,6) biochemi urea 50 mg, NaCl 20mg, MgSO4•E7H2O 20mg, cal conversion of glucose 1-phosphate to 3- CaCl•E2H2O 10mg, 1.0M phosphate buffer ketoglucose 1-phosphate by intact cells of (pH 7.0) 5.0ml and sucrose 2g in 100ml. Agrobacterium tumefaciens was reported and Cultivation was carried out at 27•Ž with was proved to be composed of the following shaking on a reciprocal shaker. The cells three processes: 1) entry of the substrate into were harvested at the middle logarithmic the cells by an active transport mechanism, growth phase (0.4•`0.8mg dry cells per one 2) conversion of the substrate to the product ml of medium), washed three times with 5 by D-glucoside 3-dehydrogenase,7) and 3) mm Tris-chloride buffer (pH 8.2), suspended exit of the product to the reaction medium in the same buffer and used as the washing by an unidentical mechanism. This paper cells. The oxidation reaction was performed deals with the biochemical conversion of in a reaction mixture having following com uridine diphosphate glucose (UDP-glucose) to position: UDP-glucose (C.F. Boehringer and uridine diphosphate 3-ketoglucose (UDP-3- Soehne GmbH Mannheim, Germany) 3ƒÊ- ketoglucose), a novel nucleotide sugar, by moles, Tris-chloride buffer (pH 8.2) 12 ƒÊmoles washing cells of Aarobacterium tumefaciens. and washed cells 7 to 8 mg (wet weight) in A strain of plant tumor-inducing bacterium, 1.0ml. Incubation was carried out at 27•Ž Agrobacteriumtumefaciens M-24 (Strk, glucose+, with shaking. At indicated intervals, an sucrose+, glucose 1-phosphate-, glucose 1- aliquot (0.5ml) of the reaction mixture was removed and centrifuged to obtain a clear 1) E. E. C. Lin, J. P. Koch, T. M. Chused and supernatant fraction. The supernatant frac S. E. Jorgensen, Proc. Natl. Acad. Sci. U.S., 48, 2145 tion was used for determination of product (1962). 2) S. Hayashi, J. P. Koch and E. E. C. Lin, J. and substrate. The amount of 3-ketoglycoside Biol. Chem., 239, 3080 (1964). was photometrically estimated by measuring 3) D. G. Fraenkel, F. Falcoz-Kelly and B. L. Horecker, Proc. Natl. Acad. Sci. U.S., 52, 1207 (1964). the absorbance at 334mƒÊ after alkali treat 4) B. M. Pogell, B. R. Maity, S. Frukin and S. ment in 0.1 N NaOH for 3 min8); UDP-3-keto Shapiro, Arch. Biochem. Biophys., 116, 406 (1966). 5) S. Fukui, J. Bacteriol., 97, 793 (1969). glucose gives a molar extinction coefficient 6) S. Fukui and S. Miyairi, ibid., 101 (1970) ap pearing in March issue. 7) K. Hayano and S. Fukui, J. Biol. Chem., 242, 8) 5. Fukui and K. Hayano, Agr. Biol. Chem., 33, 3665 (1967). 1013 (1969). 322 S. Fuxur (E334), 4.48x103 M-1cm-1 Total amount of TABLE I. PAPER ELECTROPHORESIS AND PAPER uridine derivative was also photometrically CHROMATOGRAPHY OF URIDINE DIPHOSPHATE determined by measuring the absorbance at 3-KETOGLUCOSE 262 m,ƒÊ (E262, 10.0•~103 at pH 7.0). Time course of 3-ketoglycoside formation from UDP-glucose by washed cells is shown in Fig. 1, in which termination of the oxidation was observed after 3hr incubation. During the incubation, any change in both levels of acid labile-phosphate (7min phosphate9)) and the absorbance at 262mƒÊ in the reaction mix ture was not observed. Isolation of the 3-ketoglycoside was per formed from the reaction mixture of 3hr incubation. Bacterial cells in the reaction mixture were removed by centrifugation, and Paper electrophoresis was performed by using Toyo the supernatant fraction thus obtained was -filter paper No. 51 in 40mm sodium tetraborate at treated with the resin (cation changer, pH 9.2 in a cooled pressure apparatus at 13 volts Amberlite IRC H-form). The supernatant per cm for 3 hr. Descending paper chromatography treated was neutralized with dil-NaOH, and was carried out on sheets of Toyo-filter paper No. 51 then applied to a column of activated carbon with the following solvent system: acetone, acetic acid, water (4.0:1.2:1.0). :Spots were detected by to adsorb the 3-ketoglycoside. The column molybdate reagent and by UV-absorption test under was washed with distilled water, and the 3- UV-lamp. Migration rate of compound tested was presented as a relative rate against orthophosphate. ketoglycoside adsorbed was eluted with 50% aqueous ethylalcohol. The eluate was lyophi lized and used as purified sample of the 3- ketoglycoside. The 3-ketoglycoside sample gave a single spot in both paper electrophoresis and paper chromatography (Table I). After alkali treat ment with 0.1 N NaOH,8) a characteristic ultraviolet-absorption spectrum was revealed as seen in Fig. 2. When the 3-ketoglycoside was treated with ƒ¿-D-3-ketoglucosidase10) which has high specificity toward ƒ¿-D-3-ketoglucosidic linkage, both 3-ketoglucose and uridine di- phosphate (UDP) were formed in an equi molar amount as described below. The treat FIG. 1. Time Course of Uridine Diphosphate 3- ment was carried out in a reaction mixture Ketoglucose Formation from Uridine Diphosphate containing 0.2 ƒÊmole (based on uridine moiety) Glucose by Washing Cells. of the 3-ketoglycoside, 100 umoles of phosphate 9) W. W. Umbreit, R. H. Burris and J. F. Stauffer, buffer (pH 7.0) and 0.3 unit of ƒ¿-D-3-keto- "Manometric techniques and related methods for the study of tissue metabolism," Burgess Publishing Co., 10) K. Hayano and S. Fukui, J. Bacterial., 101 Minneapolis, 1945, p. 185197. (1970) appearing in March issue. UDP-3-Ketoglucose Formation by Washing Cells of Agrobacterium tumefaciens 323 by paper chromatography in both solvent systems of acetone, acetic acid, water (4.0: 1.2:1.0) and methylethylketone, acetone, water (3.0:1.0:0.6).11) To identify uridine derivative formed, both paper electrophoresis and paper chromatography were employed as described in Table I. The reaction mixture of the 10 min incubation gave only one spot showing UV-absorption with the same mi gration rate as that of authentic sample of UDP. A fraction corresponding to UDP was eluted from the filter paper, and acid labile phosphate in the fraction was determined to be 1.04 ƒÊmole per 1.0 ƒÊmole of uridine moiety. FIG. 2. Ultraviolet-absorption Spectra of Uridine Thus, the 3-ketoglycoside was identified to be Diphosphate 3-Ketoglucose. uridine diphosphate 3-ketoglucose. In ele Concentrations: 127 mƒÊmole in 1ml of water mental analysis, UDP-3-ketoglucose gave and 109 mƒÊmole in 1ml of 0.1 N NaOH. C, 29.9%; H, 3.33% (calculated for C15H25O17N2P2Na2: C, 29.6%o; H, 3.29%). glucosidase in 1.0ml. The reaction was per Infrared-absorption spectrum of the nucleotide formed at 20•Ž in a cuvett of a spectrophoto sugar is shown in Fig. 3. meter (Hitachi Model EPU-2). Hydrolysis of The conversion of UDP-glucose to UDP-3- the 3-ketoglycoside was followed by measuring ketoglucose by washing cells was strongly in the increase in the absorbance at 310mƒÊ at hibited by 2,4-dinitrophenol, whereas glucoside which 3-ketoglucose gives E, 3.8•~103 in 0.1M 3-dehydrogenase which catalyzes the oxidation of the substrate was not affected by this in phosphate buffer (pH 7.0).8) After 10 min incubation, the amount of 3-ketoglucose that hibitor.12) This phenomenon suggests the liberated reached the maximum level, 0.19 ƒÊ- presence of active transport system(s) for mole in 1.0ml. 3-Ketoglucose was identified UDP-glucose entry and/or UDP-3-ketoglucose FIG. 3. Infrared-absorption Spectrum of Uridine Diphosphate 3-Ketoglucose. Thickness: 1.0mg in 100mg KBr (Tablet). 11) S. Fukui and R. M. Hochster, J. Amer. Chem. 12) S. Fukui, J. Biochem., 69, 873 (1969). Soc., 85, 1697 (1963). 324 S. FUKUI exit as essential factor in the biochemical cells of the bacterium,13) the physiological conversion. Recently, the existence of an significance of nucleotide 3-ketoglucose is still inducible active transport mechanism for obscure. UDP-glucose in the bacterium was demon This investigation was supported in part by a grant strated in our separate paper. l2) from the Ministry of Education. Although the conversion of adenosine di Sakuzo FUKUI phosphate glucose, cytosine diphosphate glu Institute of Applied Microbiology, cose and thymidine diphosphate glucose, University of Tokyo, Tokyo these are precursors for bacterial lipopoly- Received December 5, 1969 saccharides, to their corresponding 3-ketoglu cose derivatives was taken place by washing 13) S. Fukui, unpublished data..
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