smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e.. However, there was a
Trang 1Trehalose-phosphate synthase of Mycobacterium tuberculosis
Cloning, expression and properties of the recombinant enzyme
Y T Pan1, J D Carroll2and A D Elbein1
1
Department of Biochemistry and Molecular Biology and2Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
The trehalose-phosphate synthase (TPS) of Mycobacterium
smegmatiswas previously purified to apparent homogeneity
and several peptides from the 58 kDa protein were
sequenced Based on that sequence information, the gene for
TPS was identified in the Mycobacterium tuberculosis
genome, and the gene was cloned and expressed in
Escheri-chia coliwith a (His)6tag 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 Kmfor UDP– glucose as the glucosyl donor was 18 mM, and that for GDP–glucose was 16 mM The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Kmfor this substrate was 7 mMwhen UDP–glucose was the glucosyl donor and 4 mMwith GDP–glucose TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mMMn2+ 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 drying-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 tuberculosisis 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-glucose-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)
Trang 2purified 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) was
iden-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, and the
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-culosiscell 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
E X P E R I M E N T A L P R O C E D U R E S
Bacterial strains and culture conditions
M smegmatiswas obtained from the American Type
Cul-ture Collection (ATCC 14468) The E coli strains 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 lacIqon an F episome,
which was transferred from the E coli cloning 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)1tetracycline
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 37C
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 mMIPTG The bacterial cells were harvested by centrifugation and the pellets were suspended
in 200 lL of protein final sample buffer [PFSB: 125 mM
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-matisTPS was also used in Western blots This antibody was shown to cross-react with the recombinant M tuber-culosisTPS [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 pyrupyru-vate 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
Trang 3the 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; MnCl2, 1 lmol; 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 37C, 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
0.1M, and the reactions were heated for 10 min at 100C to
destroy any remaining sugar nucleotide Then, NaOH was
added to a final concentration of 0.15M, and samples
were again heated at 100C 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 MgCl2in 100 lL 50 mM
Tris/HCl, pH 8.0 The reactions were stopped by heating
and the following components were added: 0.15 mM
NADH, 0.25 mM phosphoenolpyruvate, 5 mM MgCl2,
and 2 lg each of pyruvate kinase and lactate dehydrogenase
in a final volume of 200 lL 50 mMHepes buffer, pH 7.0
The rate of NADH oxidation was measured at A340 as
shown in Fig 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–[3H]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 (0–0.3M) of NH4HCO3
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 NH4HCO3 The samples were
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
R E S U L T S
Cloning and expression ofM tuberculosis TPS The M tuberculosis ORF Rv3490 (otsA) is annotated in the GenBank database as a probable a-trehalose phosphate synthase 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¢-ACTCGAGAGCATATGGCTCC
GACCGTTAGC-3¢) DC 154, the upstream primer, corresponds to nucleotides 3908220–3908243 of the
M tuberculosisH37Rv genome sequence [28] The under-lined nucleotides refer to nucleotides that have been altered
to generate an upstream NdeI site The bold A 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 mMMgCl2 and at an annealing temperature of 57C The resulting PCR product was digested with NdeI and BamI, and ligated with the expression vector pET15b (Novagen), which had been linearized with the same two enzymes
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 coli strain 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 onBLASTanalysis (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
Trang 4corresponded 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-culosisTPS, 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 coli vector was incubated in 0.1 mMor 1 mM
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 mM IPTG for 4 h was
as good ( 15.8 nmolÆmin)1 of trehalose-P with UDP– glucose and 14.4 nmolÆmin)1with GDP–glucose) as 1 mM
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 mM 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 times higher (see Table 2)
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
37C 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 mM IPTG and the cells were allowed to grow for an additional 4 h The cells were isolated by centrifugation, suspended in 50 mMNaH2PO4
pH 8.0, containing 300 mM NaCl and 10 mM 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 mM imidazole in the same buffer to remove nonspecifically bound proteins The (His)6-tagged TPS was eluted with 50 mMimidazole 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 mMTris, 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 molecular mass of 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 tuberculosis (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 The GenBank
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.
Trang 5to 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:
PEPþ UDP ! pyruvate þ UTP
by the pyruvate kinase
Pyruvateþ NADH ! lactate þ NADþ
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 smegmatisTPSs 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 coli stained 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 coli stained 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.
Trang 6with 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 recombinant TPS After an incubation of 15 min, one set of incubation 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)
Trang 7formation of trehalose-P was examined ADP, at 10 mM
concentration, inhibited the formation of trehalose-P by
70% with either UDP–glucose or GDP–glucose as
substrate, but surprisingly GDP, also at 10 mM, 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 Mg2+ The
recom-binant TPS was also stimulated by a divalent cation, but
in this case Mn2+was the most active metal ion, and gave
about a twofold increase in activity at about 10 mM
Mg2+was also active, but somewhat less so than Mn2+
(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 mM) The insert presents the Lineweaver–
Burk plot of this data and shows that the Kmfor UDP–
glucose was 18 mM and that for GDP–glucose was
16 mM 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 Kmvalue for glucose-6-P of 7 mM
when UDP–glucose is the glucosyl donor, and 4 mMwith
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-[3H]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 to3H–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 0–0.3M NH4HCO3 As shown in Fig 8A, a
sharp peak of radioactivity was eluted from the column at
0.1MNH4HCO3.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 NH4HCO3was removed by repeated evaporation in the presence of triethylamine
The concentrated radioactive peak was dissolved in
50 mM 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 The assay mixtures were as described in 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 activitya(nmolÆmin)1) Native TPS activityb(nmolÆmin)1) Heparin (lg added) UDP–glucose GDP–glucose UDP–glucose GDP–glucose
a
Enzyme produced in E coli and purified.bEnzyme purified from M smegmatis.
Trang 8authentic 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
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
D I S C U S S I O N
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
30C 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 release3H–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 In profile B, 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.
Trang 9DNA sequence homology to this gene was found in the
M tuberculosisgenome, 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, as well as Arthrobacter, 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
A C K N O W L E D G E M E N T S
M tuberculosis H37Rv was kindly provided by K Eisenach, Depart-ment of Pathology, University of Arkansas for Medical Sciences Preliminary M avium and M smegmatis sequence data were obtained from the Institute for Genetic Research (TIGR) through the website at http://www.tigr.org Genome sequencing of M avium and M smeg-matis was accomplished with support from NIAID This research was supported in part by NIH grant R03-AI-43292 to ADE.
R E F E R E N C E S
1 Birch, C.C (1963) Trehaloses Adv Carbohydr Chem 18, 201– 205.
2 Elbein, A.D (1974) The metabolism of a-a- trehalose Adv Car-bohyd Chem Biochem 30, 227–256.
3 DeVirgilio, C., Hottinger, T., Dominguez, J., Boller, T & Wiem-ken, A (1994) The role of trehalose synthesis for the acquisition of thermotolerance in yeast Eur J Biuochem 219, 179–186.
4 Iwahashi, H., Obuchi, K., Fujii, S & Komatsu, Y (1997) Effect of temperature on the role of Hsp104 and trehalose in barotolerance
in Saccharomyces cerevesiae FEBS Lett 416, 1–5.
5 Hounsa, C.-G., Brandt, E.V., Trevelein, J., Hohmann, S & Prior, B.A (1998) Role of trehalose in survival of Saccharomyces cere-vesiae under osmotic stress Microbiology 144, 671–680.
6 Crowe, J.H., Crowe, L.M & Chapman, D (1984) Preservation of membranes in anhydrobiotic organisms: The role of trehalose Science 223, 701–703.
7 Benaroudj, N., D oHee, L & Goldberg, A.L (2001) Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals J Biol Chem 276, 24261–24267.
8 Singer, M.A & Lindquist, S (1998) Multiple effects on protein folding in vitro and in vivo Mol Biol 1, 639–648.
9 Elbein, A.D (1967) Carbohydrate metabolism in Streptomyces hygroscopicus Isolation and synthesis of trehalose J Bacteriol 96, 1623–1628.
10 Clegg, J.S & Evans, D.R (1961) The physiology of blood treha-lose and its function during flight in the blowfly J Exp Biol 38, 771–792.
11 Elbein, A.D & Mitchell, M (1973) Levels of glycogen and tre-halose in Mycobacterium smegmatis J Bacteriol 113, 863–873.
12 Wider, F.G., Tighe, J.J & Brennan, P.J (1972) Turnover of acyl-glucose, acyltrehalose and free trehalose during growth of Myco-bacterium smegmatis on glucose J Gen Microbiol 73, 539–546.
13 Brennan, P.J & Nikaido, H (1995) The envelope of mycobacteria Annu Rev Biochem 64, 29–63.
14 Takayama, K & Armstrong, E.L (1976) Isolation, characteriza-tion and funccharacteriza-tion of 6-mycolyl-6¢-acetyltrehalose in
Mycobacteri-um tuberculosis Biochemistry 15, 441–447.
15 Cabib, E & Leloir, L.F (1958) The biosynthesis of trehalose-phosphate J Biol Chem 231, 259–275.
16 Candy, D.J & Kilby, B.A (1958) Site and mode of trehalose biosynthesis in the locust Nature 183, 1584–1595.
17 Murphy, T.A & Wyatt, G.R (1965) The enzymes of glycogen and trehalose synthesis in silk moth fat body J Biol Chem 240, 1500– 1508.
18 Lornitzo, F.A & Goldman, D.S (1964) Purification and prop-erties of the transglucosylase inhibitor of Mycobacterium tuberculosis J Biol Chem 239, 2730–2734.
19 Roth, R & Sussman, M (1966) Trehalose synthesis in the cellular slime mold, Dictyostelium discoideum Biochim Biophys Acta 122, 225–231.
20 Pan, Y.T., Drake, R.R & Elbein, A.D (1996) Trehalose-P syn-thase of mycobacteria: Its substrate specificity is affected by polyanions Glycobiology 6, 453–461.
Trang 1021 Lapp, D., Patterson, B.W & Elbein, A.D (1971) Properties of a
trehalose-P synthase from Mycobacterium smegmatis J Biol.
Chem 246, 4567–4579.
22 Kaasen, I., Falkenberg, P., Styrvold, O.B & Strom, A (1992)
Molecular cloning and physical mapping of the otsBA genes which
encode the osmoregulatory trehalose pathway of Escherichia coli.
J Bacteriol 174, 889–898.
23 Guo, N., Puhlev, I., Brown, D.R., Mansbridge, J & Levine, F.
(2000) Trehalose expression confers dessication tolerance on
human cells Nature Biotechnol 18, 168–171.
24 Chen, Q., Ma, E., Behar, K.L., Xu, T & Haddad, C.C (2002)
Role of trehalose-P synthase in anoxia tolerance and development
in Drosophila melanogaster J Biol Chem 277, 3274–3279.
25 Gutman, P.D., Carroll, J.D., Masters, C.I & Minton, K.W.
(1994) Sequencing, targeted mutagenesis and expression of a recA
gene required for the extreme radioresistance of Deinococcus
radiodurans Gene 141, 31–37.
26 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,
Seid-mann, J.G., Smith, J.A & Struhl, K (1989) Current protocols in
molecular biology John Wiley and Sons, New York, USA.
27 Trevelyan, W.E., Procter, D.P & Harrison, J.S (1950) Detection
of sugars on paper chromatograms Nature 116, 444–445.
28 Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C.,
Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry,C.E.,
IIITekaia, F., Badcock, K., Basham, D., Brown, D.,
Chilling-worth, T., Connor, R., D avies, R., D evlin, K., Feltwell, T.,
Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K.,
Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K.,
Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter,
S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E.,
Taylor, K., Whitehead, S & Barrell, B.G (1998) Deciphering the
biology of Mycobacterium tuberculosis from the complete genome
sequence Nature 393, 537–544.
29 Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang,
Z., Miller, W & Lipman, D.J (1997) Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs.
Nucleic Acid Res 25, 3389–3402.
30 Besra, G.S., Bolton, R.C., McNeil, M.R., Ridell, M., Simpson,
K.E., Glushka, J., van Halbeek, H., Brennan, P.J & Minnikin,
D.E (1992) Structural elucidation of a novel family of
acyl-trehaloses from Mycobacterium tuberculosis Biochemistry 31,
9832–9837.
31 Besra, G.S & Brennan, P.J (1997) The mycobacterial cell
envel-ope: a target for novel drugs against tuberculosis J Pharm.
Pharmacol 49, 25–30.
32 Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H & Crowe, L.M.
(1995) Trehalose and sucrose protect both membranes and
pro-teins in intact bacteria during drying Appl Environ Microbiol 61,
3592–3597.
33 Carninici, P., Nishiyama, Y., Westover, A., Itoh, M.,
Nagaoka, S., Sasaki, N., Okazaki, Y., Muramatsu, M &
Hayashizaki, Y (1988) Thermostabilization and thermoactiva-tion of thermolabile enzymes by trehalose and its applicathermoactiva-tion for the synthesis of full-length cDNA Proc Natl Acad Sci USA 95, 520–524.
34 Wiemken, A (1990) Trehalose in yeast, stress protectant rather than reserve carbohydrate Antonie Van Leeuwenhoek 58, 209–217.
35 Matula, M., Mitchell, M & Elbein, A.D (1971) Partial purifica-tion of a highly specific trehalose-P phosphatase from Myco-bacterium smegmatis J Bacteriol 107, 208–217.
36 Winderickx, J., de Winde, J.H., Crauwels, M., Hino, A., Hoh-mann, S., van Dijck, P & Thevelein, J.M (1996) Regulation of genes encoding subunits of the trehalose synthase complex in Saccharomyces cerevesiae: Novel variations of STRE-mediated transcription control Mol Gen Genet 252, 470–482.
37 Hengge-Aronis, R., Klein, W., Lange, R., Rimmele, M & Boos, W (1991) Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in sta-tionary-phase thermotolerance in Escherichia coli J Bacteriol.
173, 7918–7924.
38 Thevelein, J.M (1984) Regulation of trehalose activity by phos-phorylation and dephosphos-phorylation during developmental transi-tions in fungi Exp Mycol 12, 1–12.
39 Vogel, G., Fiehn, O., Jean-Richard-dit-Bressel, L., Boller, T., Wiemken, A., Aeschbacher, R.A & Wingler, A (2001) Trehalose metabolism in Arabidopsis: Occurrence of trehalose and molecular cloning and characterization of trehalose-6-P synthase homo-logues J Exp Bot 52, 1817–1826.
40 Zaragoza, O., Blazquez, M.A & Gancedo, C (1998) Disruption
of the Candida albicans TPS1 gene encoding trehalose-6-phos-phate synthase impairs formation of hyphae and decreases infectivity J Bacteriol 180, 3809–3815.
41 D eSmet, K.A.L., Weston, A., Broen, I.N., Young, D B & Robertson, B.D (2000) Three pathways for trehalose biosynthesis
in mycobacteria Microbiology 146, 199–208.
42 Murata, K., Mitsuzumi, H., Nakada, T., Kubota, M., Chaen, H., Fukuda, S., Sugimoto, T & Kurimoto, M (1996) Cloning and sequencing of a cluster of genes encoding enzymes of trehalose biosynthesis from thermophilic archaebacterium Sulfolobus acid-ocaldarius Biochim Biophys Acta 1291, 177–181.
43 Murata, K., Hattori, K., Nakada, T., Kubota, M., Sugimoto, T & Kurimoto, M (1996) Cloning and sequencing of trehalose bio-synthesis genes from Arthrobaacter sp Biochim Biophys Acta
1289, 10–13.
44 Wannet, W.J.B., denCamp, H.J.M., Wisselink, H.W., van der Drift, C & van Griensven, L.D.&.Vogels, G.D (1998) Purifica-tion and characterizaPurifica-tion of trehalose phosphorylase from the commercial mushroom Agaricus bisporus Biochim Biophys Acta
1425, 177–188.
45 Rimmele, M & Boos, W (1994) Trehalose-6-phosphate hydrolase
of Escherichia coli J Bacteriol 176, 5654–5664.