Pentose Metabolism in Mycobacterium smegmatis: Specificity

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1 JOURNAL OF BATEROLOGY, Nov. 1976, p opyright American Society for Microbiology Vol. 128, No. 2 Printed in U.S.A. Pentose Metabolism in Mycobacterium smegmatis: Specificity of nduction of Pentose somerases KEN ZUMOR,* KE YAMANAKA, AND A. D. ELBEN* Department of Food Science, Kagawa University, Miki-cho, Kagawa-ken, Japan, and Department of Biochemistry, The University of Texas ealth Science enter, San Antonio, Texas 78284* Received for publication 1 June 1976 The induction of D-xylose, D-ribose, L-arabinose, and -lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. r-xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and ilyxose. -Arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas n-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases. n previous studies (6, 7), we reported the purification, crystallization, and properties of a new enzyme, D-ribose isomerase, from cells of Mycobacterium smegmatis grown on D-ribose. The r-ribose isomerase was also induced when cells were grown on L-lyxose and L-rhamnose, and all of these activities were shown to reside in the same protein (6). r-ribose isomerase activity was not found in cells of Aerobacter aerogenes or Escherichia coli grown on D-ribose, whereas D-ribokinase was induced by D-ribose in these organisms. D- Ribose isomerase activity, however, was found in cells of A. aerogenes or E. coli grown on L- rhamnose. The present report describes some experiments on the specificity of induction of the pentose isomerases in M. smegmatis. MATERALS AND METODS hemicals. Sugars and polyols were obtained from Sigma hemical o.; Trypticase soy broth was obtained from Baltimore Biological Laboratory; peptone was obtained from Kyokuto Seiyaku o., Tokyo, Japan; and yeast extract was from Oriental Kobo o., Tokyo, Japan. All other chemicals were obtained from commercial sources and used without further purification. Growth of the organism. M. smegmatis was maintained on slants on Trypticase soy agar. These slants were used to inoculate 500-ml flasks containing 100 ml of peptone (0.5%), yeast extract (0.2%), and glycerol (0.5%) medium. After 48 h of growth at 30, 100 ml of this culture was used to inoculate 5- liter flasks containing 2 liters of the same medium. ells were grown in this medium for 48 h at 30 with shaking and were harvested by centrifugation. After washing with water, cells were used for the induction experiments. To determine the ability to utilize various sugars and polyols as a sole source of carbon, the mineral salts medium of eath and Ghalambour (3) supplemented with 0.5% of various substrates was used. Slants of M. smegmatis maintained on Trypticase soy agar were used to inoculate test tubes (16 by 180 mm) containing 6 ml of the mineral salts medium. The cultures were grown at 30 with shaking, and the growth was measured in a itachi 101 spectrophotometer with a test tube adapter at 600 nm. nduction experiment. One gram of washed cells grown on the glycerol medium was suspended in the mineral salts medium containing 0.5% of the various sugars and was incubated at 35 for 6 h with shaking. After incubation, cells were harvested by centrifugation and sonicated in 10 ml of M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, p 7.5, containing 0.5 mm Mnl2. After centrifugation at 12,000 rpm for 20 min, the supernatant was used for determination of pentose isomerase activities. Assay of pentose isomerase. Pentose isomerases were assayed in reaction mixture containing the following components: 0.5 ml of 0.05 M Tris-hydrochloride buffer, p 7.4; 0.05 ml of 0.01 M Mnl2; 0.05 ml of 0.1 M various pentoses and enzyme in a final volume of 1 ml. When assays were run for D-arabinose isomerase, 0.05 M glycine buffer, p 9.3, was also tested in addition to Tris, since D-arabinose isomerase has been reported to be inhibited by Tris buffer (8, 18). The reaction was initiated by the addition of pentose, and after incubation for 10 min at 350, the reaction was terminated by the addition of 0.1 ml of 10% trichloroacetic acid. The formation of ketopentoses was determined by the cysteinecarbazole reaction (2). One unit of enzyme is defined as the amount that converts 1 umol of pentoses to respective ketopentoses in 1 min. Protein determi- 587

2 588 ZUMOR, YAMANAKA, AND ELBEN nation was made by the method of Warburg and hristian (15). RESULTS Growth of various sugars and polyols. n a previous paper (4), it was reported that the growth of M. smegmatis was much more rapid and that the lag period was shorter when D- mannose and D-fructose were used as sole carbon source rather than D-glucose. Table 1 presents data on the growth of M. smegmatis in mineral salts medium with various sugars or polyols as sole carbon source. Growth was apparent after 1 day of incubation when D-mannose, D-fructose, L-arabinose, D- lyxose, D-xylose, four pentitols, L-rhamnose, D-mannitol, D-sorbitol, and inositol were used as carbon sources. After a few days, growth of the cells was also observed in the medium containing D-glucose, D-ribose, and L-sorbose. Other sugars and polyols tested were not utilized as growth substrates by M. smegmatis under these cultivation conditions. Production of pentose isomerases by M. smegmatis under growing conditions. Five pentose isomerase activities, D-arabinose (L-fucose) isomerase, L-arabinose isomerase, D- lyxose (D-mannose) isomerase, D-ribose (irhamnose) isomerase, and D-xylose isomerase TABLE 1. J. BATEROL. were measured in cells grown on nutrient broth supplemented with 0.5% of the various sugars. ells were harvested after 20 h of growth, and pentose isomerase activities in the cells were assayed and expressed in units of enzyme per 1 g of wet cells as presented in Table 2. M. smegmatis produced four pentose isomerases which were inducible enzymes, since they were only formed when a particular sugar was included in the growth medium. No D-arabinose isomerase activity was found in any cells. L-Arabinose isomerase was produced in cells grown on L-arabinose medium as well as on D- galactose medium. D-Lyxose isomerase activity was found in the cells grown on D-mannose, D- lyxose, and D-ribose medium. D-Ribose isomerase, as described previously (6), was produced in cells grown on either D-ribose, L-rhamnose, or L-yxose medium. n contrast to the above isomerases, D-xylose isomerase was found only in the cells grown on D-xylose medium. All of the pentose isomerases except n-xylose isomerase were found in cells grown on two or three different sugars (Table 2). D-Galactose, which was not utilized by this organism as the sole carbon source, induced L-arabinose isomerase more effectively than L-arabinose. Thus, it was of interest to examine the relationship between the structure of the effective Growth of Mycobacterium smegmatis on various sugars and polyols as a sole carbon source arbon source (0.5%) Growtha b D-Glucose D-Galactose D-Mannose ±± +± Fructose ± L-Sorbose D-Arabinose - L-Arabinose - +4± + ± D-Lyxose D-Ribose D-Xylose ±± L-Xylose - - D-Fucose - L-Fucose - L-Rhamnose Dulcitol D-Mannitol D-Sorbitol )-Arabitol L-Arabitol Ribitol Xylitol nositol a -, 0 to 0.05 units of absorbancy at 600 nm; +, 0.05 to 0.10 units of absorbancy at 600 nm; +, 0.10 to 0.05 units of absorbancy at 600 nm; + +, 0.50 to 1.5 units of absorbancy at 600 nm. b ulture time (days).

3 VOL. 128, 1976 PENTOSE METABOLSM N M. SMEGMATS 589 TABLE 2. Production ofpentose isomerases by Mycobacterium smegmatis Pentose isomerasea (U/mg of protein) Sugars added (0.5%) Gram of wet p of media cells after growth nose 1)-Arabi- isom- nose L-Arabi- isom- smrlyxose D-Xylose D-Ribose erase erase Lsomerase isomerase isomerase D-Glucose D-Galactose D-Mannose D-Arabinose iarabinose D-Lyxose L-Lyxose D-Ribose D-Xylose L-Xylose None a A value of 0 indicates isomerase activity of less than U/mg of protein. inducer and that of enzyme substrate in more detail. nduction specificity of pentose isomerases by M. smegmatis. ntact cells of M. smegmatis grown on glycerol medium were used for induction experiments. The conditions of the induction, enzyme extraction, and enzyme assay are described in Materials and Methods. During this induction experiment, no growth and little change of p of the medium was observed. As shown in Table 3, the four pentose isomerases were induced by various substrates. Xylose isomerase was induced only by i-xylose. The inducers of D-ribose isomerase were 1>ribose, L-rhamnose, and ilyxose, and the configuration of carbons 1 to 3 of -ribose seemed to be essential for induction of the enzyme (Fig. 1). i1arabinose isomerase was induced by various compounds including r>-galactose, L-arabinose, L-arabitol, dulcitol, and D-fucose. Although -galactose, dulcitol, and -fucose were not utilized as carbon sources by this organism, they induced L-arabinose isomerase strongly. The configuration of carbons 2 to 4 of -arabinose seemed to be essential to induce this enzyme (Fig. 2). From the results showing that L- arabitol and dulcitol were inducers of the enzyme and that inositol could not induce the enzyme, it seems likely that the open-chain form of the inducer was effective for the enzyme induction. r.-lyxose isomerase was induced by -mannose, -lyxose, D-ribose, dulcitol, and inositol. Dulcitol was not used by M. smegmatis as a carbon source but did induce the enzyme. Thus, it is not clear from these data what is the essential structure for induction of n-lyxose isomerase (Fig. 3). TABLE 3. nducers somerase activity of cells induced by sugars and polyols Activity induceda (U/mg of protein) D-Arabi- L-Arabi- D- D-Xylose D-Ribose nose nose Lyxose.. isomer- isomer- isomer- ase ase ase ase ase D-Glucose D-Galactose i-mannose n-arabinose L-Arabinose D-Lyxose L-Lyxose 0 NDb ND ii.ribose D-Xylose L-Xylose i-fucose L-Fucose L-Rhamnose Dulcitol )-Mannitol D-Sorbitol )-Arabitol L-Arabitol Ribitol Xylitol myo-nositol None a A value of 0 indicates isomerase activity of less than U/mg of protein. ND, Not determined. DSUSSON From the induction data presented in this paper, it is clear that four pentose isomerases, D-xylose isomerase, n-ribose isomerase, L-arabinose isomerase, and n-lyxose isomerase, are induced in M. smegmatis. t seems that the inducer specificity of some of these pentose isomerases in M. smegmatis is relatively broad. D-Xylose isomerase. n M. smegmatis, D-

4 590 ZUMOR, YAMANAKA, AND ELBEN xylose isomerase was induced specifically by D- xylose as reported by many workers in various microorganisms (16). This enzyme was the only one that showed absolute specificity for inducer in this organism. D-Ribose isomerase. n our previous studies (6, 7), ii-ribose isomerase was purified and crystallized from cells of M. smegmatis grown on either z-ribose or L-rhamnose. From the comparison of the properties of these enzymes in- O l l O 2O - 1 O 2O O 3 D-Ribose L-Lyxose L-Rhamnose FG. 1. nducers ofd-ribose isomerase. fk-, Essential structure. O : i 2O O :_ 2O O ' 3 2o :_ 2O O 2O L-Arabinose D-Galactose D-Fucose L-Arabitol Dulcitol FG. 2. nducers ofl-arabinose isomerase. --, Essential structure. O2, / O \ O -- 0O D-Mannose \,O ol/ - 0,O O \O O/ - - D-Lyxose duced by ii-ribose or L-rhamnose, it was postulated that both enzymes were the same protein. The cells of E. coli or A. aerogenes grown in the presence of L-rhamnose have isomerase activity for both L-rhamnose and D-ribose. owever, when -these organisms are grown in the presence of D-ribose, they have no isomerase activity for either of these substrates (7). t has been reported by Mortlock and Wood (9) that the first enzyme in the D-ribose catabolic pathway in A. aerogenes is D-ribokinase, which is induced by D-ribose. L-Arabinose isomerase. Previously, many studies concerning the purification and properties of L-arabinose isomerases from various organisms were reported (5, 10, 12, 14, 17). The enzyme was purified and obtained as a homogeneous protein from E. coli by Patrick and Lee (12) and also isolated in crystalline form from the extracts of Lactobacillus gayoni by Nakamatu and Yamanaka (10). n all of these works, L-arabinose isomerase was induced by L-arabinose, and other sugars had no ability to induce the enzyme. As mentioned in the results, in M. smegmatis, L-arabinose isomerase was induced not only by L-arabinose but also by D-galactose, L-arabitol, and dulcitol. Sugars not utilized by the organism, D- galactose and dulcitol, still induced the enzyme. D-Lyxose isomerase. M. smegmatis has been shown to produce D-mannose isomerase which catalyzes the isomerization of z-lyxose and D- mannose when the organism is grown on D- mannose (4). As presented in this paper, this isomerase was induced not only by D-mannose but also by r-lyxose, D-ribose, dulcitol, and inositol. O2 0 \ // O D-Ri bose O,O J. BATEROL. O 20 --O 04\ O O-- / O-- - O O -O 2O nositol Dulcitol FG. 3. nducers of D-lyxose isomerase. / O\

5 VOL. 128, 1976 Palleroni and Doudoroff (11) isolated a D- mannose isomerase from Pseudomonas saccharophila grown on fructose as the sole carbon source. Takasaki (13) reported that the D-mannose isomerase was induced in the cells of Streptomyces aerocolonigenes grown on D-mannose. n addition, Anderson and Allison isolated r-lyxose isomerase from A. aerogenes grown on t-lyxose as the sole carbon source. The i-lyxose isomerase was active for isomerization of D- mannose to fructose but was not induced by growing cells on D-mannose. n M. smegmatis, pentose isomerases, except i-xylose isomerase, were induced by more than one inducer. n some cases, one inducer induced two pentose isomerases, namely D-ribose which induced D-ribose and oyxose isomerases and dulcitol which was a potent inducer of both i- arabinose and iayxose isomerases. Of the various inducers, some of them were not able to cause growth, whereas some others were not substrates for the isomerases that they induced. M. smegmatis seems to be unusual among microorganisms in that the inducer specificity of pentose isomerases is relatively broad. Work in this laboratory is now in progress to determine whether these isomerase activities induced by different sugars reside in same protein. AKNOWLEDGMENT This work was supported in part by Public ealth Service grant A from the National nstitute of Allergy and nfectious Diseases. LTERATURE TED 1. Anderson, R. L., and D. P. Allison Purification and characterization of D-lyxose isomerase. J. Biol. hem. 240: Dishe, Z., and E. Borenfreund A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. Biol. hem. 192: eath, E.., and M. A. Ghalambour Metabolism of L-fucose.. The purification and properties of L- PENTOSE METABOLSM N M. SMEGMATS 591 fuculose kinase. J. Biol. hem. 237: ey-ferguson, A., and A. D. Elbein Purification of a D-mannose isomerase from Mycobacterium smegmatis. J. Bacteriol. 101: oritsu,.,. Sasaki, and M. Tomoeda Pentose metabolism in andida utilis. V. L-arabinose ketol isomerase. Agric. Biol. hem. 34: zumori, K., M. Mitchell, and A. D. Elbein Evidence that the isomerization of i.-ribose and L-rhamnose is catalyzed by the same enzyme in Mycobacterium smegmatis. J. Bacteriol. 126: zumori, K., A. W. Rees, and A. D. Elbein Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis. J. Biol. hem. 250: zumori, K., and K. Yamanaka Purification and crystallization of D-arabinose isomerase from Aerobacter aerogenes by polyethylene glycol. Agric. Biol. hem. 38: Mortlock, R. P., and W. A. Wood Metabolism of pentoses and pentitols by Aerobacter aerogenes.. Demonstration of pentose isomerase, pentulokinase, and pentitol dehydrogenase enzyme families. J. Bacteriol. 88: Nakamatu, T., and K. Yamanaka rystallization and properties of L-arabinose isomerase from Lactobacillus gayonii. Biochim. Biophys. Acta 178: Palleroni, N. J., and M. Doudoroff Mannose isomerase of Pseudomonas saccharophila. J. Biol. hem. 218: Patrick, J. W., and N. Lee Purification and properties of an L-arabinose isomerase from Escherichia coli. J. Biol. hem. 243: Takasaki, Y Kinetic and equilibrium studies on D-mannose-D-fructose isomerization catalyzed by mannose isomerase from Streptomyces aerocolorigenes. Agric. Biol. hem. 31: Tomaeda, M.,. oritsu, and. Sasaki Pentose metabolism in andida acetobutylicum.. L-arabinose ketol isomerase. Agric. Biol. hem. 33: Warburg, O., and W. hristian solierung und Krystallization des Garungsferments. Enolase. Biochem. Z. 310: Yamanaka, K D-Xylose isomerase, p n W. A. Wood (ed.), Methods in enzymology, vol. 9. Academic Press nc., New York. 17. Yamanaka, K., and W. A. Wood L-Arabinose isomerase, p n W.A. Wood (ed.), Methods in Enzymology, vol. 9. Academic Press nc., New York. 18. Yamanaka, K., and K. zumori nhibition of D- arabinose (L-fucose) isomerase activity by Tris and its analogues. Agric. Biol. hem. 40: