Báo cáo Y học: Trehalose-phosphate synthase of Mycobacterium tuberculosis Cloning, expression and properties of the recombinant enzyme pdf

Trehalose-phosphate synthase of Mycobacterium tuberculosis Cloning, expression and properties of the recombinant enzyme Y. T. Pan 1 , J. D. Carroll 2 and A. D. Elbein 1 1 Department of Biochemistry and Molecular Biology and 2 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPSwasidentifiedintheMycobacterium tuberculosis genome, and the gene was cloned and expressed in Escheri- chia coli with a (His) 6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of  58 kDa on SDS/PAGE. Approximately 1.3 mg of puri- fied TPS were obtained from a 1-L culture of E.coli( 2.3 g cell paste). The purified recombinant enzyme showed a single band of  58 kDa on SDS/PAGE, but a molecular mass of  220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP–glucose, CDP–glucose, GDP–glucose, TDP–glucose and UDP–glucose, with ADP–glucose, GDP– glucose and UDP–glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose- P from any of the nucleoside diphosphate glucose deriva- tives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP–glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The K m for UDP– glucose as the glucosyl donor was  18 m M ,andthatfor GDP–glucose was  16 m M . The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the K m for this substrate was  7m M when UDP–glucose was the glucosyl donor and  4m M with GDP–glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 m M Mn 2+ . This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis. Trehalose is a nonreducing disaccharide in which the two glucoses are linked in an a,a-1,1-glycosidic linkage [1]. This naturally occurring anomer of trehalose is widespread in nature, being found in bacteria, fungi, yeast, plants, insects and lower animals [2]. In many organisms, trehalose synthesis is induced in response to a small set of specific environmental conditions. In particular, trehalose is accu- mulated during periods of nutrient starvation, desiccation, and after exposure to mild heat shock [3,4]. Thus, it has been proposed that this sugar serves as a stabilizer of cellular structures under stress conditions [5]. In agreement with this hypothesis, in vitro studies have shown that trehalose has an exceptional capacity for protecting biological membranes and enzymes from the adverse effects of freezing, or drying- induced dehydration [6], as well as from stress induced by exposure to oxygen radicals [7]. Trehalose may also aid in protein folding [8]. This disaccharide may also play other roles in various cells. For example, in Streptomyces hygroscopicus, very little trehalose is found in the vegetative mycelia, but trehalose is abundant in the spores [9]. Thus, in this organism and other bacteria and fungi, as well as in various insects, trehalose probably functions as a storehouse of glucose and energy, such as for flight muscle contraction, and for spore germination [10]. On the other hand, trehalose appears to be constitutively present in mycobacteria, as it is present in the cytosol at levels of 1–3% of the dry weight of these cells under normal growth conditions [11]. Although the function of this cytosolic trehalose is not known, the trehalose pool in growing and well-fed cells of Mycobacterium smegmatis is subject to rapid turnover, suggesting that the free trehalose pool is not accumulated just for storage of glucose [12]. Furthermore, trehalose is an integral component of a number of different glycolipids in mycobacteria, and these compounds appear to be essential cell wall structures [13]. In fact, one of the toxic components of the cell wall of Mycobacterium tuberculosis is called cord factor and has the structure, trehalose-6,6¢-dimycolate [14]. The transfer of glucose from UDP–glucose to glucose- 6-phosphate to form trehalose-6-phosphate was first demonstrated using cell-free extracts of Saccharomyces cerevesiae [15]. This reaction was also demonstrated in locusts [16], in silk moths [17], in M. tuberculosis [18] and in Dictyostelium discoideum [19]. The enzyme catalyzing this reaction, i.e. the trehalose-phosphate synthase (TPS), was Correspondence to: A. D. Elbein, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA. Fax: + 1 501 686 8169, Tel.: + 1 501 686 5176, E-mail: Elbeinaland@UAMS.edu Abbreviations: IPTG, isopropyl thio-b- D -galactoside; TPS, trehalose- phosphate synthase. (Received 24 May 2002, revised 6 September 2002, accepted 21 October 2002) Eur. J. Biochem. 269, 6091–6100 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03327.x purified to near homogeneity from cytosolic extracts of M. smegmatis [20], and that enzyme preparation could use any of the glucose sugar nucleotides (i.e. ADP–glucose, CDP–glucose, GDP–glucose, TDP–glucose and UDP– glucose) as glucosyl donors to form trehalose-6-P [21]. However, there was a difference in the rate of trehalose-P formation with the different glucosyl donors, with ADP– glucose, GDP–glucose and UDP–glucose being best [21]. The substrate specificities, or the other enzymatic properties, of TPSs from these other organisms have not been reported. The gene for the trehalose-P synthase (otsA)wasiden- tified in Escherichia coli [22] and has been expressed in various organisms [23,24]. Neither this enzyme, nor the yeast enzyme, have been expressed or isolated in sufficient amounts to determine the substrate specificity with regard to the glucosyl donor, or other properties of these TPSs. The enzyme from M. smegmatis was purified to apparent homogeneity and several peptides from this protein were sequenced [20]. Based on the amino acid sequences of these peptides, the putative TPS gene from M. tuberculosis was identified. In this report, we describe the cloning and expression of the M. tuberculosis tps gene in E.coli,andthe production of active TPS in these cells. The properties of the recombinant enzyme have been determined, and are com- pared to the properties of the TPS purified from M. smeg- matis. This expression system should provide sufficient amounts of recombinant TPS for complete structural characterization of this enzyme. Furthermore, trehalose is not found in any mammalian cells but it appears to play an important role as a structural component of the M. tuber- culosis cell wall, and might also function as a stabilizer and protector of membranes and proteins when this organism undergoes latency. Therefore, enzymes involved in the biosynthesis of trehalose may represent excellent target sites for new chemotherapeutic drugs against tuberculosis and other mycobacterial diseases. EXPERIMENTAL PROCEDURES Bacterial strains and culture conditions M. smegmatis was obtained from the American Type Cul- ture Collection (ATCC 14468). The E.colistrains DH5a and HMS-F [25] were used for cloning and expression studies, respectively. HMS-F is a derivative of the expres- sion strain HMS174(DE-3) (Novagen). HMS174(DE-3) contains a chromosomal isopropyl thio-b- D -galactoside (IPTG)-inducible T7 RNA pol gene. HMS-F contains an additional copy of the lac repressor lacI q on an F episome, which was transferred from the E.colicloning strain XL-1 (Stratagene). This addition effectively represses expression from the T7 promoter on the E.coli expression vector pET15b (Novagen) in the absence of IPTG. HMS-F was routinely cultured in the presence of 10 lgÆmL )1 tetracycline to maintain carriage of the F episome. E.coli strains were cultured in L-broth and on L-agar supplemented with 100 lgÆmL )1 ampicillin, 20 lgÆmL )1 kanamycin or 10 lgÆmL )1 tetracycline, individually or in combination where applicable. M. tuberculosis H37Rv was cultured in Middlebrook 7H9 broth and on Middlebrook 7H10 agar, supplemented in each case with 10% (v/v) oleic acid- albumin-dextrose complex. All bacterial strains were cul- tured at 37 °C. Materials Nucleoside diphosphate sugars, nucleoside mono-, di- and triphosphates, alkaline phosphatase, heparin and anthrone were from Sigma Chemical Co. Trypticase Soy broth was from Becton Dickinson Co. Electrochemiluminescence Western blotting detection reagents were from Amersham Pharmacia Biotech Inc. Ni–NTA HisÆbinding resin was obtained from Novagen, and used according to the manufacturer’s recommendations. LB broth was from Fisher Scientific Co. Except where otherwise specified, all DNA manipulation enzymes, including restriction endo- nucleases, polymerases and ligase, were supplied by New England Biolabs, and used according to the manufacturer’s instructions. Custom oligonucleotide primers were com- mercially synthesized by Integrated DNA Technologies (Coralville, IA, USA). All other chemicals were from reliable chemical suppliers and were of the best grade available. Western immunobloting E.colistrains containing recombinant pET15b or p996A458 were cultured for 2–4 h in L broth containing ampicillin, tetracycline and 0.1 or 1 m M IPTG. The bacterial cells were harvested by centrifugation and the pellets were suspended in 200 lL of protein final sample buffer [PFSB: 125 m M Tris/HCl, pH 6.8, 10% (v/v) glycerol, 10% (v/v) b-merca- ptoethanol, 10% (w/v) SDS, 0.25% (w/v) Bromophenol blue]. The suspensions were boiled for 10 min and centri- fuged briefly to remove any insoluble material. The supernatant liquid was subjected to PAGE and proteins were transferred to nitrocellulose as described previously [26]. Proteins with (His) 6 tags were detected with mouse anti(His) 6 -IgG (Amersham) and goat anti-mouse alkaline phosphatase conjugate (GAM-AP, Biorad Inc.), and visu- alized with commercially available colorimetric substrates (Immun-Blot, Biorad). Rabbit antibody prepared against the purified M. smeg- matis TPS was also used in Western blots. This antibody was shown to cross-react with the recombinant M. tuber- culosis TPS [20,21]. In this case, the antibody reactive bands were detected by the electrochemiluminescence Western blotting detection reagents according to the manufacturer’s protocol. Proteins were also detected by staining with Coomassie blue. Assay of TPS activity Formation of trehalose-P could be assayed by a colorimetric method which involved destroying all reducing sugars by treatment with alkali and then detection of trehalose by the anthrone method. This assay was useful for both crude extracts and for more purified enzyme preparations. Trehalose formation could also be assayed spectrophoto- metrically in an enzyme coupled assay where the UDP, released when glucose was transferred from UDP–glucose to glucose-6-P, was detected and quantified by coupling the conversion of phosphoenolpyruvate to pyruvate by pyru- vate kinase, and the conversion of pyruvate to lactate by lactate dehydrogenase. In this final reaction, the oxidation of NADH, i.e. the formation of NAD + , was measured at 340 nm. This spectrophotometric assay worked well with 6092 Y. T. Pan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the purified TPS and in fact gave identical results to the colorimetric assay (see Fig. 5), but it was not reliable with crude extracts or with partially purified preparations. It was also not reliable when GDP–glucose was used as the glucosyl donor. Incubation mixtures for measuring TPS activity by the colorimetric assay contained the following components in a final volume of 0.1 mL: nucleoside diphosphate glucose, 1 lmol; glucose-6-P, 1 lmol; MnCl 2 ,1lmol; Tris/HCl pH 8.0, 5 lmol, and an appropriate amount of enzyme. Assays were carried out in the absence or presence of 1 lg heparin per incubation mixture. Incubations were usually for 15–30 min at 37 °C, but other times were used as indicated in the text. Trehalose-6-P formation was deter- mined by a colorimetric assay as reported previously [20,21]. This procedure is briefly described here. At the end of the incubation, HCl was added to a final concentration of 0.1 M , and the reactions were heated for 10 min at 100 °Cto destroy any remaining sugar nucleotide. Then, NaOH was added to a final concentration of 0.15 M ,andsamples were again heated at 100 °C to destroy all reducing sugars. Trehalose is a nonreducing sugar and is stable to both the mild acid treatment and the alkaline treatment. It could then be determined and quantitated by the anthrone colorimetric method for hexoses. For assaying trehalose-P formation by the spectro- photometric method, assay mixtures contained the same components as in the colorimetric method, i.e. 1 lmol each of glucose-6-P, UDP–glucose and MgCl 2 in 100 lL50m M Tris/HCl, pH 8.0. The reactions were stopped by heating and the following components were added: 0.15 m M NADH, 0.25 m M phosphoenolpyruvate, 5 m M MgCl 2 , and 2 lg each of pyruvate kinase and lactate dehydrogenase in a final volume of 200 lL50m M Hepes buffer, pH 7.0. The rate of NADH oxidation was measured at A 340 as showninFig.5. In some experiments, trehalose-P formation was also measured by a radioactive assay, and this assay was used to obtain radioactive trehalose-P for characterization of the product. In these studies, assay mixtures were as described above except that UDP–[ 3 H]glucose (0.1 lCi) was used as the glucosyl donor. Reactions were stopped by the addition of acid as above, and after heating for 10 min, the reaction mixture was applied to a DE-52 ion exchange resin column. After thorough washing with water, the phosphorylated sugars were eluted with a gradient (0–0.3 M )ofNH 4 HCO 3 . An aliquot of each fraction was removed and assayed for its radioactive content. The radioactive peak was pooled and concentrated to dryness a number of times in the presence of triethylamine to remove NH 4 HCO 3 .Thesampleswere dissolved in water, adjusted to pH 8.0 with glycine buffer and treated with alkaline phosphatase to remove the phosphate group. The radioactive sugar was then identified by its migration on paper chromatograms as compared to various known sugar standards. Identification of sugars by paper chromatography Radioactive sugars were separated by chromatography on Whatman 3MM paper by streaking them over a 15-cm area of the paper. Papers were usually 23 cm in width and 45 cm long. Sugar standards (glucose maltose, trehalose, raffinose, stachyose) were spotted on the sides of the papers. Papers were chromatographed in either (A) ethyl acetate : pyri- dine : water (12/5/4, v/v/v) or (B) n-butanol : pyri- dine : water (5/3/2, v/v/v). Standard sugars were detected with the silver nitrate dip [27], and radioactive sugars were detected by cutting a strip of the paper into 0.5 cm pieces, from the origin to the solvent front, and counting each strip in a scintillation counter. RESULTS Cloning and expression of M. tuberculosis TPS The M. tuberculosis ORF Rv3490 (otsA) is annotated in the GenBank database as a Ôprobable a-trehalose phosphate synthaseÕ. The 1500-bp ORF is located at nucleotides 3908234–3909733 of the M. tuberculosis H37Rv genome. The TPS gene (otsA in E.coli) potentially encodes a 500- residue polypeptide, with a predicted molecular mass of 55 863 Da. A 1.5-kb PCR product was amplified from M. tubercu- losis H37Rv genomic DNA using the oligonucleotide primers DC 154 (5¢-ACTCGAGAGC ATATGGCTCC CTCG-3¢) and DC 155 (5¢-AGCGGGA TCCGCTT GACCGTTAGC-3¢). DC 154, the upstream primer, corresponds to nucleotides 3908220–3908243 of the M. tuberculosis H37Rv genome sequence [28]. The under- lined nucleotides refer to nucleotides that have been altered to generate an upstream NdeI site. The bold ÔAÕ represents the first nucleotide of the otsA ORF. DC 155, the down- stream primer, is complimentary to nucleotides 3909730– 3909753 of the M. tuberculosis genomic sequence, and has been altered to incorporate a downstream BamHI site (underlined nucleotides). PCR amplification was carried out with 1.5 m M MgCl 2 and at an annealing temperature of 57 °C. The resulting PCR product was digested with NdeIandBamI, and ligated with the expression vector pET15b (Novagen), which had beenlinearizedwiththesametwoenzymes. This generated the recombinant plasmid p996A458. The entire cloned (His) 6 –otsA gene fusion was sequenced to confirm the fidelity of the amplification, and p996A458 DNA was electroporated into the E.colistrain HMS-F [29]. The resulting ampicillin- and tetracycline-resistant trans- formant was cultured with and without IPTG, and expres- sion of a (His) 6 -tagged protein of the predicted size (58 kDa) was demonstrated by Western blotting (data not shown). Sequence analysis of the TPS gene ( otsA ) Based on BLAST analysis (27: http://www.ncbi.nlm.nih.gov/ BLAST) of the predicted M. tuberculosis otsA amino acid sequence, otsA exhibits amino acid sequence homology to a number of trehalose-P synthases from prokaryotic and eukaryotic sources, including M. leprae (77% identity, 6% similarity), Candida albicans (36% identity, 17% similarity), Aspergillus niger (34% identity, 16% similarity), Saccharo- myces cerevisiae (38% identity, 53% similarity), Arabidopsis thaliana (34% identity, 15% similarity) and Escherichia coli (32% identity, 44% similarity). In addition, M. avium contains an otsA homolog which is 80% identical and 9% similar to M. tuberculosis otsA. TBLASTN comparison with the unfinished M. smegmatis genome sequence being completed by TIGR detected a coding sequence that Ó FEBS 2002 Trehalose synthesis in mycobacteria (Eur. J. Biochem. 269) 6093 corresponded to a polypeptide with 74% identity and 6% similarity. Fig. 1 shows the predicted amino acid sequence of the M. tuberculosis TPS and its sequence alignment with those of homologous ORFs from several other mycobac- teria and OtsA of E.coli, as indicated by CLUSTALW alignment. The alignment shows several regions of the protein with very high homology, for example the sequence of 20 amino acids starting at position 397 of the M. tuber- culosis TPS, and another sequence of about 16 amino acids starting at position 421 of the M. tuberculosis TPS. Isolation and purification of recombinant TPS To maximize conditions for production of recombinant TPS, the E.colivector was incubated in 0.1 m M or 1 m M IPTG for 4 h or overnight, and then cells were harvested and disrupted by sonication. The cytosolic fraction was then assayed for TPS enzymatic activity, using either GDP–glucose or UDP–glucose as the glucosyl donor. Enzyme assays indicated that 0.1 m M IPTG for 4 h was as good ( 15.8 nmolÆmin )1 of trehalose-P with UDP– glucose and 14.4 nmolÆmin )1 with GDP–glucose) as 1 m M IPTG for 4 h in inducing the formation of TPS. In addition, 4 h of incubation with ITPG gave a somewhat higher yield of TPS activity than did an overnight incubation in IPTG (data not shown). Thus, cells were routinely induced in 0.1 m M IPTG for 4 h. This experi- ment also demonstrated that the recombinant enzyme preparation had almost equal activity with either UDP– glucose or GDP–glucose, both in the presence or absence of heparin. In previous studies with extracts from M. smegmatis, the activity of the native TPS with UDP– glucose (0.3 nmolÆmin )1 ) was the same as that for GDP– glucose in the absence of heparin, but in the presence of heparin, activity with UDP–glucose was as much as five timeshigher(seeTable2). The recombinant TPS having a (His) 6 tag could be purified on a nickel column as has been widely used for various recombinant proteins (Novagen). TPS col- onies were grown in 500 mL LB medium containing 100 lgÆmL )1 ampicillin and 10 lgÆmL )1 tetracycline at 37 °C until the optical density at 600 nm reached a value of 0.6. At this time, TPS fusion protein synthesis was induced by the addition of 0.1 m M IPTG and the cells were allowed to grow for an additional 4 h. The cells were isolated by centrifugation, suspended in 50 m M NaH 2 PO 4 pH 8.0, containing 300 m M NaCl and 10 m M imidazole, and disrupted by sonication. The cell debris were removed by centrifugation and the supernatant fraction was used as the crude extract for isolation of TPS. The extract was applied to a Ni–NTA column of His resin to bind the fusion protein, and the column was washed extensively with 30 m M imidazole in the same buffer to remove nonspecifically bound proteins. The (His) 6 -tagged TPS was eluted with 50 m M imidazole in the same buffer. This fraction was concentrated to a small volume on an Amicon concentrator to remove salts and then diluted  20-fold with 50 m M Tris, pH 7.5. This fraction was used as the source of purified TPS and was subjected to SDS/ PAGE. As shown in Fig. 2A lane 2, a single band with a molecularmassof 58 kDa was detected in the elution fraction. This protein was also detected by Western blot- ting (Fig. 2B, lane 1) using the antibody prepared against the M. smegmatis TPS. From a 1-L culture of the trans- fected E.coli( 2.3 g cell paste),  1.3 mg purified TPS was obtained. The purified TPS showed a single protein band of  58 kDa on SDS gels either when stained with Coomassie blue or when subjected to Western blotting using antibody prepared against the M. smegmatis TPS. However, when the purified recombinant protein was subjected to gel filtration on a calibrated column of Sephracryl S-300, the major peak of TPS enzymatic activity emerged in the region suggesting a molecular weight of  220 kDa (Fig. 3). These data suggest that the active TPS probably exists as a tetramer. Properties of the recombinant TPS The activity of the purified recombinant TPS showed a linear increase with increasing protein concentration, from 5 Fig. 1. CLUSTALW alignment of M. tu berculos is (Mt) OtsA predicted amino acid sequence with those of homologous ORFs from M. avium (Ma), M. smegmatis (Ms) and E. coli (Ec). The M. avium and M. smegmatis homologs were identified by searching the respective unfinished TIGR genome sequences with TBLASTN .TheGenBank accession number for the E.coli sequence is NP-416410. Perfectly conserved residues are indicated (*), conservative and semiconservative substitutions are indicated (:) and (.), respectively. Gaps introduced by CLUSTAL to optimize the alignment (-) are also indicated. 6094 Y. T. Pan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 to 20 lg of protein per incubation, as demonstrated in Fig. 4B. The increase in enzymatic activity was also linear with time of incubation for about 20 min and then began to level off (Fig. 4A). This figure also shows that the activity with GDP–glucose as the glucosyl donor was equal to or slightly higher than activity with UDP–glucose. In these experiments, trehalose-P formation was determined by the colorimetric method, but the formation of trehalose-P from UDP–glucose could also be measured by a spectrophoto- metric assay in which the production of UDP was coupled to the oxidation of NADH by utilizing the following two reactions in a coupled assay: PEP þ UDP ! pyruvate þ UTP by the pyruvate kinase Pyruvate þ NADH ! lactate þ NAD þ by the lactate dehydrogenase As shown in Fig. 5, when the purified TPS was used in these reactions, the colorimetric assay and the spectrophotometric assay gave almost identical results in terms of the effect of enzyme concentration on formation of trehalose-P. Thus in this figure, the amount of trehalose-P formed (nmol) was measured by the anthrone assay (colorimetric), and was compared to the amount of NAD + produced (nmol) in the coupled assay, as an indirect measure of the amount of trehalose-P synthesized. However, for most of the studies described here, the anthrone assay was used. The substrate specificity of the recombinant enzyme for the nucleoside diphosphate glucose substrate was examined, as shown in Table 1. The data demonstrates that the recombinant enzyme was most active with the purine sugar nucleotides, ADP–glucose and GDP–glucose, whereas the pyrimidine nucleotides were somewhat less effective. UDP– glucose was the best of the pyrimidine nucleotides and slightly less effective than either ADP–glucose or GDP– glucose, but TDP–glucose and CDP–glucose were signifi- cantly less effective. Thus, the M. tuberculosis and the M. smegmatis TPSs are rather unusual glucosyltransferases, as most enzymes of this class are fairly specific for both the base portion of the nucleoside diphosphate sugar, as well as for the sugar component. The recombinant enzyme showed somewhat better activity with GDP–glucose over UDP–glucose, whereas the M. smegmatis TPS displayed better activity with UDP– glucose than with GDP–glucose when heparin was added to the enzyme assays. Therefore, we compared the effect of heparin on trehalose-P formation from UDP–glucose or GDP–glucose with the purified recombinant TPS, as well as with the partially purified enzyme from M. smegmatis. Table 2 shows that heparin did stimulate the formation of trehalose-P from both UDP–glucose and GDP–glucose Fig. 2. SDS/PAGE of recombinant TPS. (A) Profiles of proteins from recombinant E.colistained with Coomassie blue. Lane 1, molecular marker proteins; lane 2, purified TPS; lane 3, crude extract. (B) Western blots of protein fractions from transfected E.colistained with antibody prepared against the purified M. smegmatis TPS. Lane 1, Purified recombinant TPS; lane 2, crude recombinant E.coli. Fig. 3. Elution profile of TPS on Sephacryl S-300. Recombinant TPS was placed on a column of Sephacryl S-300 (1.2 · 110 cm), and the column was eluted with 10 m M Tris, pH 7.5. Fractions were collected and assayed for TPS activity. Molecular weight markers were also run on this column and their elution position is shown by the various arrows: b-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; BSA, 66 kDa; carbonic anhydrase, 29 kDa. Ó FEBS 2002 Trehalose synthesis in mycobacteria (Eur. J. Biochem. 269) 6095 with the recombinant, but the activation was much lower (about a twofold increase) than that observed with the TPS isolated from M. smegmatis (about a fivefold increase). We do not know why the recombinant enzyme differs in regard to heparin activation from the wild-type TPS. It is possible that the His tag either alters the protein conformation in such a way as to prevent the interaction of heparin with the enzyme, or the positively charged His tag binds the polyanion and blocks its interaction. This latter possibility seems unlikely, as increasing the amount of heparin in the incubation, as shown in Table 2, did not change the degree of stimulation. Hopefully, future studies comparing the structure of the recombinant protein to that of the native TPS will answer this question. The specificity of recombinant TPS for the glucosyl acceptor, i.e. sugar-6-phosphate, was also examined. As observed in previous studies with the TPS purified from M. smegmatis, glucose-6-P was active as a glucosyl acceptor with both UDP–glucose and GDP–glucose, and GDP–glucose was a somewhat better glucosyl donor than UDP–glucose. However, glucose-6-P could not be replaced by either mannose-6-P, fructose-6-P or glucos- amine-6-P, when any of the glucose sugar nucleotides were used as glucosyl donors (data not shown). Thus both the recombinant TPS, as well as the wild-type mycobacterial TPS, are specific for the glucosyl acceptor but not for the glucosyl donor. Since the formation of trehalose-P from the nucleoside diphosphate glucose (i.e. UDP–glucose or GDP–glucose) produces a nucleoside diphosphate, i.e. UDP or GDP, the effect of various nucleoside diphosphates on the Fig. 4. Effect of time and protein concentration on TPS activity. (A) Assay mixtures were as described in the text with either UDP–glucose or GDP–glucose and 2 lg TPS. At the times shown in (A), an aliquot of the incubation mixture was removed and assayed for its trehalose content. (B) Assay mixtures contained different amounts of TPS as indicated in the figure, and incubations were for 15 min. The amount of trehalose produced in each incubation was determined as described in Experimental procedures. Fig. 5. Comparison of the colorimetric assay for measuring trehalose-P formation with the coupled enzymatic assay. Incubation mixtures for trehalose-P formation were the same for both assays and are described in Experimental procedures but contained various amounts of the recombinantTPS.Afteranincubationof15min,onesetofincubation mixtures was assayed by the anthrone colorimetric method and the other set was assayed by the coupled enzyme assay where the amount of UDP produced was measured by its conversion of PEP to pyruvate and pyruvate to lactate. The formation of NAD + in this second reaction was measured spectrophotometrically. Table 1. Substrate specificity of TPS for nucleoside diphosphate glu- cose. One lmol of each nucleotide was added to the standard incu- bation mixture described in Experimental procedures. Glucose nucleotide TPS activity (nmolÆmin )1 ) ADP–glucose 6.3 CDP–glucose 4.3 GDP–glucose 6.1 TDP–glucose 3.3 UDP–glucose 5.6 6096 Y. T. Pan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 formation of trehalose-P was examined. ADP, at 10 m M concentration, inhibited the formation of trehalose-P by  70% with either UDP–glucose or GDP–glucose as substrate, but surprisingly GDP, also at 10 m M , only inhibited the reaction with UDP–glucose ( 50%) but not with GDP–glucose. In addition, UDP did not inhibit either reaction. Although the TPS did not show an absolute require- ment for a cation, activity was previously shown to be stimulated by divalent cations such as Mg 2+ . The recom- binant TPS was also stimulated by a divalent cation, but in this case Mn 2+ was the most active metal ion, and gave about a twofold increase in activity at about 10 m M . Mg 2+ was also active, but somewhat less so than Mn 2+ (data not shown). Kinetic constants for TPS The effect of substrate concentration on the activity of the TPS was determined as shown in Figs 6 and 7. Fig. 6 shows the effect of increasing concentrations of either UDP– glucose or GDP–glucose in the presence of saturating levels of glucose-6-P (20 m M ). The insert presents the Lineweaver– Burk plot of this data and shows that the K m for UDP– glucose was  18 m M and that for GDP–glucose was  16 m M . The concentration of glucose-6-P for half maxi- mal velocity was also measured at saturating concentrations of either GDP–glucose or UDP–glucose as shown in Fig. 7. Again the insert demonstrates the Lineweaver–Burk plot of this data and indicates a K m value for glucose-6-P of 7 m M when UDP–glucose is the glucosyl donor, and 4 m M with GDP–glucose as substrate. Characterization of the product To characterize the product synthesized by the recombinant enzyme, incubations were set up as described in Experi- mental procedures, but they contained UDP-[ 3 H]glucose rather than the unlabeled substrate. The reaction was stopped with HCl, and the mixture was heated as described in methods to hydrolyze UDP–glucose to 3 H–glucose. The incubation mixture was applied to a DE-52 column to bind phosphorylated sugars, and after thorough washing with water, the phosphorylated sugars were eluted using a gradient of 0–0.3 M NH 4 HCO 3 .AsshowninFig.8A,a sharp peak of radioactivity was eluted from the column at  0.1 M NH 4 HCO 3. This migration pattern is very similar to that shown by other sugar phosphates such as glucose- 6-P. The fractions containing radioactivity were pooled and concentrated, and the NH 4 HCO 3 was removed by repeated evaporation in the presence of triethylamine. The concentrated radioactive peak was dissolved in 50 m M glycine buffer pH 8.5 and treated overnight with alkaline phosphatase to release the phosphate group from the sugar. The incubation mixtures were deionized with mixed-bed ion-exchange resin to remove salt and the neutral sugar solution was streaked on Whatman 3MM paper and separated by chromatography in solvent A to identify the sugars. Fig. 8B shows the radioactive profile on these papers, and indicates that only one radioactive band was detected that migrated about 35 cm from the origin and had the same migration position as authentic trehalose. This radioactive band was clearly separated from maltose and glucose. This radioactive product also migrated with Fig. 6. Effect of concentration of glucosyl donor (UDP–glucose or GDP–glucose) on TPS activity. Theassaymixtureswereasdescribedin Experimental procedures, but contained various amounts of the sub- strates UDP–glucose or GDP–glucose as indicated. TPS activity is expressed as the amount of trehalose-P synthesized in nmolÆmin )1 .The insert shows the Lineweaver–Burk plot of the data. Table 2. Comparison of the effect of heparin on recombinant and native TPS activity. Recombinant TPS activity a (nmolÆmin )1 ) Native TPS activity b (nmolÆmin )1 ) Heparin (lg added) UDP–glucose GDP–glucose UDP–glucose GDP–glucose 0 2.7 4.6 2.4 5.0 1 4.7 6.0 11.4 7.6 5 4.4 4.4 10.8 7.7 10 3.9 6.3 10.0 8.0 20 4.0 5.9 9.2 7.7 a Enzyme produced in E. coli and purified. b Enzyme purified from M. smegmatis. Ó FEBS 2002 Trehalose synthesis in mycobacteria (Eur. J. Biochem. 269) 6097 authentic trehalose in solvent B and was a nonreducing sugar based on its lack of reactivity in the reducing sugar test and its resistance to alkaline degradation (data not shown). These data indicate that the recombinant enzyme is a trehalose-P synthase and that the product is trehalose- phosphate. Trehalose was also identified as the only product when GDP–glucose was used as the substrate rather then UDP–glucose. DISCUSSION Trehalose is an important sugar in mycobacteria because it serves as a component of a number of cell wall glycolipids of M. tuberculosis, including cord factor which is trehalose- dimycolate. Cord factor is an important structural compo- nent in these organisms [30], and may also serve as a donor of mycolic acids to the arabinogalactan [31]. In addition, there is increasing evidence, at least in yeast and some other organisms, to indicate that free trehalose may function in a protective capacity, and prevent these cells from suffering the adverse effects of desiccation [32], heat stress [33], freezing [34], anoxia [6,7], and so on. Thus, the reactions involved in the synthesis of trehalose-P and/or free trehalose appear to have an important and probably essential function in the physiology of many organisms. The most widely demonstrated pathway for the synthesis of trehalose involves two enzymes that catalyze the follow- ing reactions: the TPS transfers a glucose from UDP– glucose (or another glucose nucleotide) to glucose-6-P to form trehalose-P plus UDP (or another nucleoside) [21]; then trehalose-P phosphatase removes the phosphate group of trehalose-P to produce free trehalose [35]. TPS has been demonstrated in a number of different organisms including yeast [15,36], bacteria [8,22,37], fungi [19,38], insects [16,17] and plants [39]. However, the specific role, or roles, of trehalose in these various organisms has not been definit- ively established. Both copies of the gene encoding TPS were disrupted in Candida albicans and this mutant did not accumulate trehalose at stationary phase or after heat shock. Disruption of this gene did impair development of hyphae and did decrease the infectivity of the organism, but it was not lethal. Thus, the rate of growth of the mutant at 30 °C was indistinguishable from the growth rate of the wild-type, although differences between the two were noted at higher temperatures [40]. Recently, two other pathways of trehalose synthesis have been reported in bacteria. One of these pathways, demon- strated in Pimelobacter species, involves the conversion of maltose to trehalose by an intramolecular transglucosyla- tion [41]. This enzyme, called trehalose synthase, is coded for by the treS gene, which codes for a 573-amino acid protein. Interestingly, the 220 amino terminal residues were homologous to those of maltases from the yeast S. carls- bergenesis and the mosquito, Aedes aegypti. About 40% Fig. 8. Identification of the product produced by recombinant TPS. Incubation mixtures containing UDP–[ 3 H]glucose and other compo- nents were as described in Experimental procedures. After incubation, the reaction mixtures were acidified and heated to release 3 H–glucose from UDP–glucose. The mixtures were then run on a DE-52 column to bind sugar-phosphates and the column was washed exhaustively with water. The charged sugars were then eluted with a 0–0.3 M linear gradient of NH 4 HCO 3. As shown in profile A, a sharp symmetrical peak of radioactivity emerged at  0.1–0.15 M NH 4 HCO 3 and frac- tions [23–32] containing radioactivity were pooled and concentrated several times with triethylamine to remove the NH 4 HCO 3 .InprofileB, the charged compound was treated with alkaline phosphatase and subjected to paper chromatography in solvent A. The radioactive peak migrated in the same position as authentic trehalose (T) and was separated from maltose (M) and glucose (G). Fig. 7. Effect of glucose-6-P concentration on TPS activity. Assay mixtures were as described in Experimental procedures, but varying amounts of glucose-6-P were used as indicated. Both UDP–glucose and GDP–glucose were present at saturating concentrations (50 m M ). TPS activity is expressed as in Fig. 6. The insert shows the Line- weaver–Burk plot of the data. 6098 Y. T. Pan et al. (Eur. J. Biochem. 269) Ó FEBS 2002 DNA sequence homology to this gene was found in the M. tuberculosis genome, and these workers presented some evidence from cell-free studies in mycobacteria to suggest that maltose was converted to trehalose [41]. Another pathway of trehalose biosynthesis has also been found in bacteria and involves three genes (treZ, treX and treY)that encode enzymes that convert sugars from glycogen into trehalose [42]. These genes have been found in Sulfolobus acidocaldarius,aswellasArthrobacter, Brevibacterium and Rhizobium [43], and they code for a maltooligosyltrehalose hydrolase, glycogen debranching enzyme, and maltooligosyl trehalose synthase. These genes show  40% homology to regions in the genome of M. tuberculosis [41]. However, whether either of these two pathways are actually utilized for trehalose formation in mycobacteria is not known, nor is there any information on the relative contribution of these various pathways to the production of trehalose in myco- bacteria, or in other organisms. It is possible that one of these pathways could provide the trehalose for one function, while another pathway is utilized to produce trehalose for another role. In that regard, it should be noted that TPS produces trehalose-6-P whereas these other pathways pro- duce free trehalose. There are two other reactions that could give rise to trehalose. In the mushroom, Agaricus bisporus, the enzyme trehalose phosphorylase catalyzes the reversible reaction: trehalose þ Pi , glucose þ glucose-1-phosphate This phosphorolysis can result in the formation of trehalose from glucose and glucose-1-phosphate [44]. The native enzyme has a molecular mass of 240 kDa and consists of four identical 61-kDa subunits. The enzyme is highly specific for the four substrates shown in the reaction. It seems likely that under the appropriate conditions in the cell, the enzyme can catalyze either the synthesis or the degradation of trehalose. Another unusual enzyme in E.coli is the trehalose-6-P hydrolase. This enzyme is probably involved in uptake of trehalose as trehalose is transported into these cells by the phosphotransferase system which forms trehalose-6-P [45]. Based on the results with these various systems, it appears that trehalose or trehalose-P may be produced via a number of different pathways. Which pathway gives rise to which function of trehalose is not clear. For example, in mycobac- teria there is biochemical or genetic evidence for three different pathways. Is it possible that one pathway provides the trehalose for cell wall synthesis, whereas another pathway gives rise to trehalose that serves as a stabilizer of cells, or as a storehouse of glucose for energy? It is still too early in our knowledge of these various pathways to make this determination, but further characterization of the genes involved in these pathways may provide evidence as to which pathway is necessary for cells to tolerate adverse conditions, or to make complete cell wall structures to protect themselves from toxic agents. As trehalose synthesis may be essential for survival of these organisms but does not occur in mammalian cells, the pathway(s) of trehalose biosynthesis represents a potential target site for chemotherapy against tuberculosis. Once the genes and their products have been identified in these cells, deletion experiments can be performed to determine which, if any, of these reactions are essential to survival of these organisms. ACKNOWLEDGEMENTS M. tuberculosis H37Rv was kindly provided by K. Eisenach, Depart- ment of Pathology, University of Arkansas for Medical Sciences. 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Carroll 2 and. assayed by the anthrone colorimetric method and the other set was assayed by the coupled enzyme assay where the amount of UDP produced was measured by its