VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1209 9 Ramel, A., E. Stellwagen, and H. K. Schachman, Federation Proc., 20, 387 (1961). 10 Markus, G., A. L. Grossberg, and D. Pressman, Arch. Biochem. Biophys., 96, 63 (1962). "1 For preparation of anti-Xp antisera, see Nisonoff, A., and D. Pressman, J. Immunol., 80, 417 (1958) and idem., 83, 138 (1959). 12 For preparation of anti-Ap antisera, see Grossberg, A. L., and D. Pressman, J. Am. Chem. Soc., 82, 5478 (1960). 13 For preparation of anti-Rp antisera, see Pressman, D. and L. A. Sternberger, J. Immunol., 66, 609 (1951), and Grossberg, A. L., G. Radzimski, and D. Pressman, Biochemistry, 1, 391 (1962). 14 Smithies, O., Biochem. J., 71, 585 (1959). 15 Poulik, M. D., Biochim. et Biophysica Acta., 44, 390 (1960). 16 Edelman, G. M., and M. D. Poulik, J. Exp. Med., 113, 861 (1961). 17 Breinl, F., and F. Haurowitz, Z. Physiol. Chem., 192, 45 (1930). 18 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940). 19 Pressman, D., and 0. Roholt, these PROCEEDINGS, 47, 1606 (1961). THE ENZYMATIC SYNTHESIS OF GUANOSINE DIPHOSPHATE COLITOSE BY A MUTANT STRAIN OF ESCHERICHIA COLI* BY EDWARD C. HEATHt AND ALAN D. ELBEINT RACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BACTERIOLOGY, THE UNIVERSITY OF MICHIGAN Communicated by J. L. Oncley, May 10, 1962 We have previously reported' the isolation of guanosine diphosphate colitose (GDP-colitose* GDP-3,6-dideoxy-L-galactose) from Escherichia coli 0111-B4; only 2.5 umoles of this sugar nucleotide were isolated from 1 kilogram of cells. Studies on the biosynthesis of colitose with extracts of this organism indicated that GDP-mannose was a precursor;2 however, the enzymatically formed colitose was isolated from a high-molecular weight substance and attempts to isolate the sus- pected intermediate, GDP-colitose, were unsuccessful. We now wish to report the enzymatic synthesis of GDP-colitose from GDP-man- nose (Fig. 1) using extracts of a mutant strain derived from E. coli 0111-B4. This mutant (designated E. col; J-5) was isolated from aged cultures of E. coli 0111-B4, and appears to have properties similar to those of the mutant strains of Salmonella typhi-murium and Salmonella enteritidis previously reported.3' 20 Thus. E. coli J-5 exhibits the following characteristics: (1) inability to ferment galactose, (2) galactose sensitivity, (3) accumulation of uridine diphosphate galactose (when growth media contain galactose), (4) accumulation of GDP-colitose. In addition, analysis of the cell-wall lipopolysaccharide isolated from the parent and mutant organisms agreed with these findings. Thus, the parent organism produces lipo- polysaccharide containing glucose, galactose, and co'itose.4 When the mutant is grown in the absence of galactose, the lipopolysaccharide contains no galactose and little or no colitose; supplementation of the growth medium with galactose yields lipopolysaccharide that appears similar to the normal product. Materials and Methods.-Tyvelose was prepared by mild acid hydrolysis of the cell-wall lipo- polysaccharide of Salmonella typhi4' followed by neutralization, deionization, and chromatog- raphy. 3-Deoxy-D-ribohexose was a generous gift of N. K. Richtmyer, National Institutes of Health, Bethesda, Md. Uniformly labeled L-fucose was kindly provided by H. S. Isbell of the Downloaded by guest on October 1, 2021 1210 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S. CH2OH National Bureau of Standards. All o o other chemicals were obtained from CH3 commercial sources. --PPRG -- H o- PPRG Guanosine diphosphate D-glucose H was a generous gift from D. M. Carlson of this laboratory. GDP- GDP-MANNOSE GDP- COLI/OSE mannose and guanosine diphos- GUANOSINE DIPHOSPHATE GUANOSINE DIPHOSPHATE phate L-fucose (GDP-fucose) were a- D- MANNOPYRANOSIDE P(?)- 3,6-DIDEOXY-L -GALACTOPYRANOSIDE chemically prepared by condensing FIG. 1.-The conversion of GDP-mannose to GDP- the corresponding hexose 1-phos- colitose. phate with GMP morpholidate.6 Mannose 1-phosphate was prepared by the method of Posternak and Rosselet.7 The synthesis of L-fucose 1-phosphate has not been previously described. In the present studies, L-fucose was converted to L-fucose 1-phosphate by a series of procedures analo- gous to those used for the preparation of mannose 1-phosphate; thus, fucose - (crystalline) tetra- acetyl-fucose (crystalline) 1-chloro-triacetyl L-fucose L 1-diphenyl-phosphoryl-triacetyl-L- fucoside- L-fucose 1-phosphate. Although the anomeric configuration of L-fucose 1-phosphate and therefore of GDP-fucose is not known with certainty, it is assumed to be the ,B-L-pyrano- side. Thus, when 1-chlorotriacetyl-L-fucoside was converted to methyl-2,3,4-triacetyl-L-fuCopy - ranoside, only the ,8-glycoside (mp 96° could be isolated; this derivative is readily distinguish- able8 from the a-anomer (mp 670). Chromatographic solvent systems used in these studies were as follows: I. Ethanol: 1 A ammonium acetate, pH 7.4 (7:3); II. Isobutyric acid: ammonium hydroxide: water (57:4:39); III. 0.1 M phosphate, pH 6.8: ammonium sulfate: n-propanol (100: 60:2); IV. Ethyl acetate: acetic acid:water (3:1:3); V. n-Butanol:pyridine:water (6:4:3); VI. n-Butanol:ethanol: water (10:4:3); VII. n-Butanol:pyridine:0.1 N hydrochloric acid (5:3:2); VIII. n-Butanol: acetic acid:water (4:1:5). For the determination of radioactivity on paper chromatograms, guide strips were scanned for radioactivity in a windowless 47r scanner. For quantitation, appropriate areas of the paper were cut out, suspended in a toluene solvent,9 and counted in a Packard liquid scintillation spec- trometer. Phosphate was determined by the method of Fiske and Subbarow;"° anthrone reagent' was used for the estimation of hexose; diphenylamine reagent" was used for the estimation of deoxy- ribose; dideoxyhexose was determined by the thiobarbituric acid procedure." Due to the extreme acid-lability of GDP-colitose (see Fig. 3), the acid conditions employed in the thiobarbituric acid test caused considerable hydrolysis of the nucleotide even at 37°. It was therefore necessary to perform the periodate oxidation at pH 7. Under these conditions, free colitose exhibited approximately one half the molar absorbancy index as that observed under standard conditions. Conversion of GDP-Mannose to GDP-Colitose.-E. coli J-5 was grown in Trypti- case Soy broth at 370 for 12 hr with shaking. Cells were harvested by centrifuga- tion and washed with cold 0.15 M KC1. Extracts were prepared by suspending cells in 3 volumes of water, sonicating for 5 to 10 min, and centrifuging at 25,000 X g for 30 min. The incubation mixture contained the following (/umoles in a final volume of 36.5 ml): GDP-mannose-C"4 (34,100 cpm//Amole), 12; TPN, 50; glu- cose 6-phosphate, 50; potassium fluoride, 500; Tris buffer, pH 7.2, 5,000; and 20 ml of crude extract. Disappearance of GDP-mannose and appearance of a prod- uct were followed by removing 0.4 ml aliquots at the indicated times (Fig. 2) and transferring to 1 ml of warm ethanol to stop the reactions. After cooling and centri- fuging, the supernatant fluids were concentrated in vacuo to 0.2 ml, applied to What- man 3MM paper in one inch bands, and chromatographed in solvent I. After developing the chromatograms for 17 hr, the areas of each strip corresponding to Downloaded by guest on October 1, 2021 VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1211 GDP-mannose (Rf = 0.16) and 40 ' ' GDP-colitose (Rf = 0.27) were cut A out and their radioactive content 32 determined as described. These re- N suits (Fig. 2) indicated that GDP- x° 242 mannose was rapidly converted to E A GDP-Colifose a compound with chromatographic 6 GDP-Mannose properties similar to GDP-colitose. In addition, 85 to 100 per cent of / \ the total radioactivitywasaccounted 8 1 for in these two areas of the chro- u matograms for each aliquot tested, 20 40 60 80 100 120 indicating that GDP-colitose formed MINUTES by these extracts is not further FIG. 2.-The rate of enzymatic conversion of GDP-mannose to GDP-colitose. The details of the metabolized under these conditions.conditions experiment are described in the text. After 120-min incubation, the re- mainder of the mixture was added to 100 ml of warm ethanol and centrifuged, and the supernatant fluids were chromatographed in the same manner as described for the aliquots. The radioactive band was eluted with water and rechromato- graphed on Schleicher and Schuell 589-Blue Ribbon paper first in solvent II and then in solvent I. A single radioactive band was observed in each case which cor- responded in mobility to GDP-colitose (isolated from E. coli J-5). Using these pu- rification procedures, 6.6 umoles of GDP-colitose were isolated. Homogeneity of the Product.-The nucleotide appeared homogeneous (on What- man 1 paper) in solvents I (Rf = 0.35), II (Rf = 0.37), and III (Rf = 0.36) and by paper electrophoresis in 0.05 M potassium phosphate buffer, pH 7.5, and in 0.05 M citrate buffer, pH 4.6; in each case, the radioactive and ultraviolet-light- absorbing spots coincided. The chromatographic and electrophoretic mobilities of the enzymatically synthesized material were identical in all instances with those of GDP-colitose which had been isolated from cells. Characterization of GDP-Colitose.-The nucleotide exhibited ultraviolet-light- absorption spectra at pH 1, 7, and 11 which were indistinguishable from authentic GDP. Analysis of the nucleotide gave the following results (molar ratios): guano- sine, 1.00; phosphorus, 2.04; 3,6-dideoxyhexose, 1.04. Inorganic phosphorus was not detected in the preparation. Guanosine was estimated from the absorbancy of the sample at 252 mAt at pH 7, assuming a molar absorbancy index of 13.7 X 103.