METABOLISM OF L-RHAMNOSE BY ESCHERICHIA COLI

METABOLISM OF L-RHAMNOSE BY ESCHERICHIA COLI I. L- RHAMNOSE ISOMERASE DOROTHY M. WILSON1 AND SAM AJL Department of Bacteriology, Walter Reed Army Institute of Research, Washington, D. C. The methyl pentose, L-rhamnose, is known to be fermented by a number of bacteria, but little information is available concerning the metabolic steps involved in this fermentation. An indication of the pathway of rhamnose metabolism was provided by the finding of Kluyver and Schnellen (1937) that propylene glycol was a major product in the fermentation by Bacillus rhamnosifermentans. Since 5 per cent of the rhamnose carbon could be accounted for as propylene glycol, they suggested that the rhamnose molecule was split into glyceraldehyde and lactaldehyde, the latter being then reduced to propylene glycol. However, it is only recently that other steps in rhamnose fermentation have been investigated. Preliminary work (Wilson and Ajl, 1955) indicated that the initial step in the metabolism of L-rhamnose by Escherichia coli involved the conversion of the methyl pentose to its corresponding ketose (L-rhamnulose or L-rhamnoketose). Tecce et al. (1955) with E. coli and Englesberg (1956) with Pasteurella pestis came independently to the same conclusion. The initial step in the metabolism of another methyl pentose, L-fucose, likewise involves the conversion of the aldose to the corresponding ketose, as shown by the work of Green and Cohen (1955, 1956) with a mutant strain of E. coli. This communication describes detailed experiments on the conversion of L-rhamnose to L- rhamnulose by E. coli grown in the presence of rhamnose. METHODS Growth conditions. Escherichia coli strain B was maintained on trypticase soy agar slants. Cultures were stored in the cold (4 C) and transferred approximately once a month. Large amounts of 1 This investigation represents a portion of a thesis submitted in partial fulfillment of the requirements for a Ph.D. degree, George Washington University, Washington, D. C. Received for publication September 24, 1956 bacterial cells were obtained by growing them in a liquid medium containing KH2PO4,.8 per cent; (NH4)2SO4,.4 per cent; yeast extract (Difco),.5 per cent; and L-rhamnose,.2 per cent at an initial ph of 7.. The cells were grown at room temperature for 16 to 2 hr with constant aeration. Preparation of acetone powders. The organisms were sedimented in a Sharples centrifuge, washed twice with distilled water, and resuspended in a small volume of water. This suspension was poured into 5 to 1 vol of acetone previously chilled at -7 C. The mixture was stirred intermittently for approximately 5 min and subsequently filtered through a Buchner funnel and washed with ether. The acetone powders thus prepared were stored in vacuo over calcium chloride at 4 C. Such preparations retained their enzymatic activity for many months. Preparation of enzyme extracts. Crude enzyme preparations were obtained by extracting 3 mg of acetone powder with 1 ml of distilled water. The suspension was then centrifuged and the cell debris discarded. The supernatant fluid was dialyzed against distilled water for 3 to 4 hr in the cold and used immediately. Unless otherwise stated, all experiments were performed in Warburg flasks in.25 M bicarbonate buffer at 33 C in an atmosphere of 95 per cent N2 and 5 per cent CO2 (final ph 7.6). Chromatographic procedures. At the end of the incubation period, reaction mixtures were deproteinized with 1,4 the volume of 5 per cent trichloracetic acid (TCA), centrifuged, and the precipitated protein discarded. Supernatant fluids were extracted twice with 2 to 3 vol of ether to remove the TCA. Appropriate aliquots of the solutions when then applied to Whatman No. 1 filter paper and chromatographed in the following solvents: (1) n-butanol :ethanol :water (5:1:4); (2) n-butanol :pyridine :water (6:4:3); and (3) n-butanol saturated with water. The benzidine spray of Bacon and Edelman (1951) 41

19571 L-RHAMNOSE METABOLISM BY E. COLI. I 411 was employed to visualize sugar spots on the paper. The purification of relatively large amounts of biologically produced rhamnulose was accomplished by applying the solution in a long line at one end of a filter paper and running the chromatogram in the n-butanol: water solvent. The bands of sugars were located by spraying narrow strips cut from each lateral margin of the paper. The unsprayed portion of the paper, corresponding to the ketose, was cut out and eluted with distilled water. Preparation of L-rhamnulose and L-fuculose. L-Rhamnulose was prepared by refluxing 3 g of L-rhamnose with 25 ml of pyridine for 3 hr. The pyridine was removed by vacuum distillalation, leaving a thick syrup consisting of a mixture of rhamnose and rhamnulose. Since difficulty was experienced in removing sufficient of the unconverted rhamnose by crystallization, the separation of the two sugars was finally accomplished by paper chromatography using n-butanol saturated with water as the solvent. The ketose was detected, eluted from the paper and dried in vacuo. Subsequent chromatographic analysis showed this material to be free of rhamnose. The osazone of this synthetic rhamnulose gave the same melting point (18 to 182 C) as the osazone prepared from an authentic sample of rhamnose. L-Fuculose was prepared from L- fucose in the same general manner. The ketose in this case was likewise separated from the aldose by paper chromatography, but the final material still contained a small quantity of an unknown sugar incompletely separated on the chromatogram. Analytical methods. Methyl pentose was determined by the method of Dische and Shettles (1948). The 1-min boiling time was used and the tubes were read after 2 hr. Ketose estimations were performed by the cysteine-carbazole method of Dische and Borenfreund (1951). When protein was present in the samples, it was precipitated by the addition of }4 the vol of 5 per cent TCA before either of these tests were run. Removal of the TCA was unnecessary with the dilutions of the samples used in the tests. RESULTS Washed cell suspensions of E. coli strain B, grown in the presence of L-rhamnose, fermented this methyl pentose with the production of acid and small amounts of gas. The reaction proceeded rapidly without any lag period. Cells grown in a similar medium with the addition of glucose instead of rhamnose did not ferment the latter in a period of 2 hr. The oxidation of rhamnose by glucose-grown cells gave a typical adaptive type of curve as shown in figure 1. In order to study the initial steps in the fermentation of rhamnose it was desirable to employ cell-free preparations of the bacteria. Water extracts of acetone powders of rhamnose-adapted cells were therefore used in all subsequent experiments. Incubation of L-rhamnose with such extracts under anaerobic conditions resulted in the formation of a ketose as measured by the cysteinecarbazole test. Readings were taken at 545 m/, at which wavelength maximum absorption of the colored product was obtained. A control tube containing rhamnose and boiled extract did not produce the ketose. The results of a typical experiment are shown in figure 2. When the identity of the ketose was determined in later experiments, the point on this curve reached at the end of the experiment was shown to correspond to approximately 35 per cent change of rhamnose to the ketose. Deproteinized samples of the reaction mixtures were chromatographed with each of the three solvent systems listed in the section on Methods. The presence of a second sugar, migrating further than rhamnose in these solvents, was detected. Development of the chromatograms with the 4-3 _ GLUCOSE 1 RHAMNOSE o 2 wz~~~~ 6 12 18 MINUTES Figure 1. Adaptation of Escherichia coli to the oxidation of rhamnose. Warburg flasks contained 1 ml of.5 M phosphate buffer (ph 7.),.5 ml of a 1 per cent suspension of E. coli grown in the presence of glucose,.5 ml of.1 M substrate, and.3 ml of NaOH in center well. Total volume 2.3 ml. Gas phase, air. Temperature 33 C.

412 WILSON AND AJL [VOL. 73 benzidine spray produced a distinct brown color in the case of rhamnose; the new sugar appeared as a clear yellow spot. The biologically produced ketose was purified chromatographically and compared with a numx.5 E too.4 z w.3 c.)v a..2.1. 2 4 6 8 I MINUTES Figure 2. The production of a ketose from rhamnose by extracts of Escherichia coli. Ketose was determined by the cysteine-carbazole test. Flasks contained 1 ml of extract,.3 ml of.2 M L-rhamnose, 1.5 ml of.5 M NaHCO3, and.2 ml of water. Gas phase, 5 per cent CO2 and 95 per cent N2. Temperature, 33 C. Sugar Fructose... Fucose... Ribulose... Xylulose... Rhamnose... Rhamnulose (synthetic)... Rhamnulose (experimental)... Fuculose... TABLE 1 Chromatography of sugars Solvent Mixture Butanol: ethanol: water Rf.26.27.29.33.4.41.41 Butanol: pyridine: water Rf.41.57.6.61.67.67.67 Color with Benzidine Spray Yellow Red-brown Yellow ber of known sugars. In table 1 are shown the Rf values of rhamnose, rhamnulose, and some other sugars in two solvent systems, as well as the colors of these sugars when sprayed with benzidine. Synthetic rhamnulose and the biologically produced sugar had similar Rf values in both solvent systems and produced the same yellow color when developed with benzidine. A mixture of these two sugars yielded a single spot on the chromatograms. Although synthetic fuculose had a similar Rf value, this sugar could be distinguished from rhamnulose by the orange color that the former produced with benzidine. Of the ketoses tested, therefore, only rhamnulose behaved in the same manner as the biologically produced ketose. A further comparison between the experimentally produced ketose and authentic rhamnulose showed that both exhibited identical absorption spectra in the cysteine-carbazole reaction (figure 3). The color that was developed in this test was reddish purple with a maximum absorption at 545 m,u. Full color development was reached in approximately 1 f hr. In order to determine whether the biologically produced ketose retained the properties of a methyl pentose, its absorption spectrum in the methyl pentose test was determined (figure 4). The results in this test with rhamnose and synthetic rhamnulose are given for comparison. It is clear that the absorption spectra of all three compounds are similar with a common peak at -J 4 U. X-X SYNTHETIC RHAMNULOSE - EXPERIMENTALLY PRODUCED RHAMNULOSE 4 45 5 55 6 65 A Yellow Figure S. Absorption spectra of synthetic and Orange experimentally produced rhamnulose as they were obtained in the cysteine-carbazole test.

19571 L-RHAMNOSE METABOLISM BY E. COLI. I 413 4 mu. The sugars were tested with and without the addition of cysteine. No reaction was obtained in the absence of cysteine, thus ruling out the possibility of a nonspecific material giving the color with sulfuric acid. Rhamnulose gave approximately 3 per cent more color in the methyl pentose test than did an equivalent amount of rhamnose. Pentoses and hexoses were negative in this reaction. It was noted in connection with these tests that the ratio of the reading in the ketose test to the reading in the methyl pentose test for L-rhamnulose was 1.11. The corresponding value for the biologically produced ketose was 1.1. In addition, both compounds gave positive tests for reducing sugar and were negative in the orcinol test for pentoses. From the results of the chromatography and of the tests given above it appeared that the biologically produced ketose was rhamnulose. Further evidence in support of this identification was obtained by the reversal of the reaction when synthetic L-rhamnulose was used as the substrate. Figure 5 shows a chromatogram of the final reaction products after incubation of the enzyme system with each of the three ketoses, L-rhamnulose, D-rhamnulose, and L-fuculose. Of the two z w CX a-.4.31.22 O.1 AnL _O BIOLOGICAL KETOSE (RHAMNULOSE)... SYNTHETIC RHAMNULOSE - KNOWN I RHAMNOSE?I U.U- ----- -1 3 35 4 45 5 I' Figure 4. Absorption spectra obtained in the methyl pentose test with synthetic rhamnulose, biologically produced rhamnulose, and rhamnose. I Z 3 LI S Figure 6. Chromatogram showing the formation of the corresponding aldoses from L-rhamnulose and L-fuculose. (1) L-Fucose marker; (2) Final L-fUCUlose reaction mixture; (8) Final D-rhamnulose reaction mixture; (4) Final L-rhamnulose reaction mixture; (5) L-Rhamnose marker. Solvent, n-butanol: ethanol: water. Descending flow for 3 hr. Paper sprayed with benzidine. rhamnulose isomers only the L form gave rise to rhamnose; the D isomer was completely inactive. This finding suggested that the experimentally formed rhamnulose was not the D form. L-Fuculose, although active in this system, gave rise to fucose, no rhamnose being detectable. This indicated that interconversion of fuculose and rhamnulose was unlikely in this system. The specificity of the water extracts used in these experiments with respect to their isomerase activity was tested with a number of sugars. Negative results were obtained with D-glucose, D-galactose, D-mannose, D- and L-XYlose, L-arabinose, and D-lyxose. A positive reaction for ketose formation was noted with L-fUcose and D-arabinose. It should be pointed out, however, that these latter conversions wvere obtained also with acetone powders prepared from cells grown on a yeast extract medium without the addition of rhamnose. L-Rhamnose isomerase, on the other hand, was present only when the cells had been exposed to rhamnose and it is therefore possible to conclude that the enzyme responsible for the

414 WILSON AND AJL [VOL. 73 conversion of L-fucose and D-arabinose to their respective ketoses is distinct from the L-rhamnose isomerase. CONCLUSIONS The experiments reported here indicate that the initial step in the metabolism of L-rhamnose by Escherichia coli strain B is the conversion of the aldose to its corresponding ketose, L-rhamnulose. The identity of the latter has been established chromatographically and by a variety of chemical tests, using authentic samples of L-rhamnulose for comparison. The new enzyme, designated as L-rhamnose isomerase, is probably adaptive since it is produced by this organism only when it is grown in the presence of rhamnose. The enzyme reacts with L-rhamnose but not with a variety of other sugars including D-mannose. The latter observations differentiate this enzyme from D-mannose isomerase described by Palleroni and Doudoroff (1956) which is capable of acting on D-rhamnose but not the L isomer. ACKNOWLEDGMENT The authors wish to thank Dr. J. K. N. Jones for a sample of D-rhamnulose, and Dr. B. L. Horecker for samples of ribulose and xylulose. SUMMARY A new enzyme, L-rhamnose isomerase, has been found in Escherichia coli adapted to the utilization of L-rhamnose. It catalyzes the reaction between L-rhamnose and L-rhamnulose. The reaction is reversible. The forward reaction proceeds until approximately 35 per cent rhamnulose is formed. REFERENCES BACON, J. S. D. AND EDELMAN, J. 1951 The carbohydrates of the Jerusalem artichoke and other compositae. Biochem. J. (London), 48, 114-126. DISCHE, Z. AND SHETTLES, L. B. 1948 A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem., 175, 595-63. DISCHE, Z. AND BORENFREUND, E. 1951 A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. Biol. Chem., 192, 583-587. ENGLESBERG, E. 1956 Gain of two enzymes concerned with rhamnose utilization by a single mutational event. Federation Proc., 15, 586. GREEN, M. AND COHEN, S. S. 1955 The enzymatic conversion of L-fucose to L-fuculose. Federation Proc., 14, 127-128. GREEN, M. AND COHEN, S. S. 1956 The enzymatic conversion of L-fucose to L-fuculose. J. Biol. Chem., 19, 557-568. KLUYVER, A. J. AND SCHNELLEN, C.: 1937 tyber die Vergarung von Rhamnose. Enzymologia, 4, 7-12. PALLERONI, N. J. AND DOUDOROFF, M. 1956 Mannose isomerase of Pseudomonas saccharophilia. J. Biol. Chem., 218, 535-548. TECCE, G., Di GIROLAMO, M., AND LAZZARI, G. F. 1955 Metabolismo del ramnosio de parte dell' Escherichia coli. Proc. 3rd Intern. Congr. Biochem., 96. WILSON, D. M. AND AJL, S. 1955 The metabolism of L-rhamnose by Escherichia coli. Biochim. et Biophys. Acta, 17, 289.