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INTRODUCTION |
Trehalose is an
,
-1,1-linked disaccharide of
D-glucose and is found in various plants, insects, and
microorganisms. In these organisms, it is believed to serve diverse
roles, one of which is as a storehouse of glucose for energy and/or for
the synthesis of cell components, another is for protection of cells
against environmental pressures such as desiccation and freezing, and for stabilization of proteins against denaturation (1). In addition to
these several functions for trehalose that have been demonstrated in
yeast, fungi, and insects, some other organisms such as mycobacteria
and corynebacteria also utilize trehalose as a structural component in
their cell walls (2). Thus, in these organisms, trehalose serves as the
backbone of several different glycolipids, such as trehalose mycolate,
various sulfolipids, and other acylated-trehalose compounds (3-5).
Because trehalose is neither synthesized by, nor utilized in, mammalian
cells, but is probably an essential molecule for the structural
integrity of the mycobacterial cell wall, the reactions involved in the biosynthesis of trehalose and trehalose glycolipids should represent excellent potential target sites for chemotherapy against
Mycobacterium tuberculosis, and other mycobacterial
pathogens. Thus, the mechanisms of action and the properties of the
enzymes involved in the biosynthesis of trehalose represent important
information for the design and development of new antimycobacterial drugs.
The major pathway for the synthesis of trehalose and trehalose
derivatives in most organisms involves the transfer of glucose from
UDP-glucose (or GDP-glucose) to glucose-6-P to produce trehalose 6-phosphate plus UDP (or GDP) (6). This reaction is catalyzed by
trehalose-P synthase (TPS),1
but this enzyme apparently differs from one organism to another in
terms of the nucleoside diphosphate glucose that is able to serve as
the glucosyl donor (7-9). For example, the TPS of Mycobacterium smegmatis and M. tuberculosis can utilize either
GDP-glucose or UDP-glucose (or even other glucose nucleotides) as
glucosyl donors for trehalose synthesis (10). On the other hand, the
TPS from Saccharomyces cerevisiae is apparently specific for
UDP-glucose (11), although there is one report of an
ADP-glucose-dependent trehalose synthase in these yeast
(12).
Organisms that utilize TPS for trehalose synthesis also contain a
phosphatase that converts trehalose-P to free trehalose (6). Although
the gene (otsB) for this enzyme has been identified in
Escherichia coli (13, 14) and other organisms, the
specificity and properties of the phosphatase have not been described
in any detail. We previously reported on the presence of TPP in
M. smegmatis (15), and this phosphatase appeared to be
relatively specific for trehalose-P, with fairly low activity toward
glucose-6-P or other sugar phosphates. Here we describe the biochemical
properties of TPP.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Culture Conditions
M. smegmatis was obtained from the American Type
Culture Collection (ATCC 14468). M. smegmatis
mc2155 was provided by Dr. W. R. Jacobs Jr., Albert
Einstein College of Medicine, New York. The E. coli strains
DH5
and HMS-F (16) were used for cloning and expression studies,
respectively. HMS-F is a derivative of the expression strain
HMS174(DE-3) (Novagen). HMS174(DE3) contains a chromosomal
IPTG-inducible T7 RNA pol gene. HMS-F contains an additional
copy of the lac repressor lacIq on an
F episome, which was transferred from 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 µg/ml tetracycline to maintain
carriage of the F episome. E. coli strains were cultured in
L broth and on L agar supplemented with 100 µg/ml ampicillin, 20 µg/ml kanamycin, or 10 µg/ml tetracycline, individually or in
combination, where applicable. M. smegmatis was
cultured in Middlebrook 7H9 broth and on Middlebrook 7H10 agar,
supplemented in each case with the 10% (v/v) oleic
acid-albumin-dextrose complex. All bacterial strains were
cultured at 37 °C.
Reagents and Materials
Trehalose phosphate, buffers, DEAE-cellulose, and other
chromatographic resins and materials were from Sigma. Sephadex G-75 and
all electrophoresis materials were from Bio-Rad. Except where otherwise
specified, all DNA manipulation enzymes, including restriction endonucleases, polymerases, and ligase, were supplied by New England Biolabs (Beverly, MA), and used according to the manufacturer's instructions. Custom oligonucleotide primers were commercially synthesized by Integrated DNA Technologies (Coralville, IA). PCR reagents were supplied by Applied Biosystems (Foster City, CA). All
other reagents were from reliable chemical companies, and were of the
best grade available. Moenomycin was a generous gift from Aventis
Pharma Deutschland GmbH and diumycin was kindly supplied by Bristol
Myers Squibb Pharmaceutical Research Institute.
Assay of the Trehalose-P Phosphatase Activity
The enzymatic activity of TPP was measured by determining the
release of inorganic phosphate from trehalose-6-P, or other sugar
phosphates. Briefly, the assay was done in a final volume of 100 µl,
containing the following components: 1 mM trehalose-6-P, 2 mM MgCl2, 50 mM Tris-HCl buffer, pH
7.5, and an appropriate amount of enzyme. After an incubation at
37 °C of 1 to 30 min, two volumes of a filtered solution containing
0.15% malachite green, 1% ammonium molybdate, and 12.5% (v/v)
concentrated HCl were added to the incubation, and the mixture was
allowed to incubate for an additional 2 min to allow for development of
color. The absorbance of the mixture was read at 630 nm and compared
with a standard solution of inorganic phosphate treated in the same manner (15).
Purification of the Trehalose-P Phosphatase
Growth and Harvesting of Bacteria--
M. smegmatis
was grown in 2-liter flasks containing 1 liter of trypticase soy broth.
One ml of an overnight culture of this organism was used to inoculate
these flasks and they were incubated at 37 °C for 24-36 h, with
shaking. Cells were harvested by centrifugation, washed with
phosphate-buffered saline, and stored as a paste in aluminum foil at
20 °C, until used.
Step 1: Preparation of Crude Extract--
All purification steps
were done at 4 °C, unless otherwise specified. Forty grams of wet
cell paste of M. smegmatis were suspended in 200 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM
2-mercaptoethanol. The cells were broken by subjecting the cell
suspension to two 3-min pulses of sonication with a Braun probe
sonicator at 80% of maximum setting. The broken cell suspension was
then centrifuged at 40,000 × g for 15 min to remove
unbroken cells and cellular debris, and the resulting supernatant
liquid, representing the cytosolic fraction, was removed and saved.
Step 2: Ammonium Sulfate Fractionation--
Solid
(NH4)2SO4 was added slowly with
stirring to the ice-cold crude extract to a final concentration of 40%
saturation, and the mixture was allowed to stir in the cold for 15 min.
The precipitate was removed by centrifugation and discarded, and solid
(NH4)2SO4 was added to the
supernatant fraction with stirring to give a final concentration of
75% saturation. The mixture was allowed to stand on ice for 15 min,
and the precipitate was isolated by centrifugation and resuspended in
40 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM 2-mercaptoethanol.
Step 3: Heat Inactivation--
Small aliquots of the above
ammonium sulfate fraction were heated at 60 °C for 45 s. The
tubes were cooled in an ice bath for 10 min, and the precipitated
protein was removed by centrifugation at 100,000 × g
for 1 h. The supernatant liquids were pooled and concentrated with
an Amicon Filtration Apparatus using a Millipore YM-10 membrane.
Step 4: Gel Filtration on Sephadex G-75 and Sephacryl
S-300--
A 3-ml aliquot of the supernatant liquid from Step 3 was
applied to a 2.6 × 95-cm column of Sephadex G-75 that had been
equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing
5 mM 2-mercaptoethanol and 1 M KCl (Buffer A).
Five-ml fractions of the eluate were collected and assayed for TPP
activity, as described above. Active fractions were pooled and
concentrated on the Amicon apparatus. This fraction was then applied to
a Sephacryl S-300 (1.6 × 90 cm) column equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM
2-mercaptoethanol and 10% glycerol (Buffer B). The column was eluted
with the same buffer and 5-ml fractions were collected and assayed for
TPP activity. Active fractions were pooled and used in subsequent steps.
Step 5: DE-52 Cellulose Chromatography--
The enzyme from the
Sephacryl S-300 column was applied to a 1.4 × 8-cm column of
DE-52 that had been equilibrated with Buffer B. After application of
the sample, the column was washed with the same buffer, containing 50 mM NaCl. The enzyme was then eluted with 90 mM
NaCl in the same buffer. Fractions were collected and those containing
active enzyme were pooled and concentrated to 3 ml on the Amicon
concentrator. The concentrated enzyme was desalted on an Econo-Pac 10 DG chromatography column that had been equilibrated with 25 mM piperizine-HCl buffer, pH 5.5, containing 5 mM 2-mercaptoethanol and 10% glycerol (Buffer C). This
buffer was also used to elute the enzyme.
Step 6: Chromatofocusing--
The enzyme from the above step was
applied to a 0.9 × 14-cm column of Polybuffer exchange (PBE 94)
that had been equilibrated with Buffer C. The enzyme was eluted from
the column with Polybuffer 74-HCl, pH 4.0, containing 5 mM
2-mercaptoethanol and 10% glycerol. Fractions of 2 ml were collected
and each fraction was analyzed for protein, enzymatic activity, and pH.
The enzyme emerged from the column at pH 4.8 to 4.4. Active fractions
were pooled, and concentrated on the Amicon apparatus.
Step 7: Native Polyacrylamide Gel
Electrophoresis--
Preparative polyacrylamide gel electrophoresis
was done at 4 °C in tubes containing 7% acrylamide (17). During
electrophoresis, the voltage was maintained at 300. One gel was stained
with Coomassie Blue stain to detect protein, whereas other gels were
cut into 2-mm sections and protein was eluted by overnight diffusion at 4 °C into Buffer B. Elutions were assayed for TPP activity.
Table I presents a summary of the purification procedure, including changes in specific activity, amount of purification, and yield of
enzyme at each step of the procedure.
Other Methods
Protein was measured with the Bio-Rad protein reagent (Bio-Rad)
using bovine serum albumin as the standard. The molecular weight of TPP
was estimated by gel filtration on Sephacryl S-300, as well as by gel
electrophoresis. Molecular weight standards included
-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa),
carbonic anhydrase (29 kDa), and cytochrome c (12 kDa).
Sequence Analysis
Open reading frames (ORFs) were identified by BLASTP alignment
with predicted amino acid sequences on GenBankTM. Multiple
amino acid alignments were performed using on line CLUSTALW alignment
program at a web site maintained by the European Bioinformatics
Institute (EMBL-EBI; www.ebi.ac.uk/clustalw/). Basic sequence analysis,
including identification of restriction sites, translations, and DNA
sequence alignments, were performed using the "Genejockey" program
(Biosoft, Cambridge, United Kingdom).
Circular Dichroism (CD) Measurements
To investigate the conformation of the recombinant TPP, near-UV
and far-UV were recorded at 25 °C with a Jasco Model 715 spectrophotometer. Protein concentrations of 1 and 0.1 mg/ml,
respectively, in 50 mM phosphate buffer, pH 7.4, were used
for recording near-UV and far-UV spectra. The reported spectra are the
average of five scans, which were smoothed and corrected for buffer
blanks. CD data were expressed as mean residue molar ellipticity with
115 as the mean residue molecular weight. Secondary structure
parameters were estimated by the computer program PROSEC derived by
Yang et al. (17).
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RESULTS |
Purification of the M. smegmatis TPP--
TPP was purified about
1600-fold to near homogeneity using the procedure described under
"Experimental Procedures," and outlined in Table
I. Two of the best steps in the
purification are shown in Fig. 1. In Fig.
1A, the ammonium sulfate fraction (40 to 75% saturation)
was applied to a column of Sephadex G-75. Because the TPP is a small
protein (27 kDa), it was readily separated from much of the protein in
the extract by gel filtration, and the activity eluted in a symmetrical
peak after the major protein peak. Fig. 1B shows the
chromatofocusing step that also gave a sharp peak of TPP activity that
was separated from much of the protein in the DE-52 eluate. At the
final stage of purification, the enzyme fraction showed a major protein
band of 27 kDa, and several minor bands on SDS gels. This purification
procedure gave a yield of enzyme of only about 1%, but that provided a
sufficient amount of protein to isolate peptides and obtain amino acid
sequence data to use for cloning the gene, and expressing recombinant
TPP.

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Fig. 1.
A, gel filtration of TPP on
Sephadex G-75. The supernatant fraction from the heat inactivation step
was applied to a 2.6 × 95-cm column of Sephadex G-75 and eluted
with 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM 2-mercaptoethanol and 1 mM KCl. Five-ml
fractions were collected and assayed for TPP activity (solid
squares) and protein (solid diamonds). B,
purification of TPP by chromatofocusing. The active fraction from DE-52
was applied to a 0.9 × 14-cm column of Polybuffer exchange as
described under "Experimental Procedures." Enzyme was eluted with
Polybuffer 74-HCl, pH 4.0, containing 5 mM
2-mercaptoethanol and 10% glycerol. Two-ml fractions were collected
and assayed for TPP activity (squares) and protein content
(diamonds).
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The major 27-kDa protein band was cut from the SDS gels and sent to
Harvard Microchemical Systems Inc. for protein sequencing by mass
spectrometry. Based on the amino acid sequence information, several
primers were prepared and used in PCR as described below.
Cloning and Sequencing of M. smegmatis TPP--
The TIGR
unfinished M. smegmatis genome sequence was screened using
the TBLASTN program for DNA sequences corresponding to the amino acid
sequences obtained from purified M. smegmatis TPP. All of
the primary amino acid sequences aligned with a region of Contig 3312. However, because this region of the genome sequence has not yet been
corrected or edited, we were unable to identify the boundaries of the
ORF corresponding to the amino acid sequence. We used oligonucleotide
primers DC172 (TCATCGCGGCGAGGTCGGCGACCGTA, complementary to nucleotides
1111068-1111096 of M. smegmatis contig 3312) and DC173
(CGAGCGCATCTTCGACGCGGCCAAGC, corresponding to nucleotides
1108256-1108281 of M. smegmatis contig 3312) to PCR amplify
a 2.8-kb region of the M. smegmatis genome that contained the putative TPP coding region. The PCR product was ligated into the
vector pCR4-TOPO (Invitrogen), generating the plasmid p996A481. Sequence analysis of the p996A481 insert demonstrated the presence of 2 complete ORFs and 1 partial ORF. ORF1 extended from 384 to 1121. The
predicted 245-residue product was highly homologous (90% amino acid
identity plus 6% similarity) with the M. tuberculosis ORF
Rv3574, which is annotated as a possible transcriptional regulator of
the Tet/AcrR family.
ORF2 was located immediately downstream of ORF1, extending
from 1122 to 1874. This potentially encoded a 250-residue 27-kDa polypeptide. BLASTP analysis of the ORF2 amino acid sequence indicated homology with trehalose-6-P phosphatases from Corynebacterium glutamicum (31% identity, 13% similarity, E = 7e
17), and from E. coli (33% identity, 11%
similarity, E = 1e
14). Lesser degrees of
homology were detected between ORF2 and the putative M. tuberculosis putative trehalose-P phosphatases Rv2006 (otsB, 28% identity, 12% similarity, = 6e
07)
and Rv3372 (otsB2, 28% identity, 13% similarity,
E = e
05). ORF3 was orientated in
opposition to ORF1/ORF2 extended from 2838 to 1875. This encoded a 35.2 kDa 328-residue with a high degree of homology (73% identity
plus 10% similarity) with M. tuberculosis ORF Rv3575c, a
possible transcriptional regulator related to the E. coli
raffinose repressor.
ORF2 (otsB) was PCR amplified using the primers DC186
(GGGGCATATGCAGCGCGC, corresponding to
nucleotides 1136-1153 of the p996A481 insert sequence) and DC187
(CGGGGATCCACGTTCTGGAGAC, complementary to
nucleotides 1915-1936 of the p996A481 insert sequence). These contained the requisite base mismatches (indicated by underlines) to
generate an upstream CATATG NdeI site and a downstream
BamHI site. The central "ATG" in DC186 represents the
start codon of the recombinant ORF. The 0.8-kb DC186/DC187 PCR product
was amplified, digested with NdeI and BamHI, and
ligated with the NdeI- and BamHI-digested expression plasmid pET15b (Invitrogen), generating the plasmid p996A504. The identity of the PCR product and the fidelity of the
amplification was confirmed by DNA sequencing. p996A504 was transformed
into the E. coli expression strain HMS-F. The predicted amino acid sequence of M. smegmatis TPP is presented in Fig.
2 and is compared with similar enzymes in
other organisms.

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Fig. 2.
CLUSTALW alignment of M. smegmatis
OtsB predicted amino acid sequence with those of homologous ORFs
from Mycobacterium leprae and
M. tuberculosis. The M. leprae homolog was
identified by searching the Wellcome Trust Sanger Institute
M. leprae genome sequence
(www.sanger.ac.uk/Projects/M_leprae/) with TBLASTN. The M. tuberculosis H37Rv ORFs Rv2006 (otsB; accession number
Z74025) and Rv3372 (0tsB2; accession number AL009198) are
annotated as possible trehalose-phosphate phosphatases. The 1327 residue Rv2006 sequence is truncated at residue 588 for the purposes of
brevity. Perfectly conserved residues are indicated by the
asterisk (*), conservative and semi-conservative
substitutions are indicated by colons (:) and
dots (.), respectively. Gaps introduced by CLUSTAL to
optimize the alignment are indicated by . Ms, Ml,
Mt3372, and Mt2006 refer to M. smegmatis, M. leprae, M. tuberculosis Rv3372, and M. tuberculosis
Rv2006, respectively.
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Preparation of Recombinant TPS--
The E. coli
expression strain HMS-F transformed with p996A504 was grown and induced
as described under "Experimental Procedures." The crude sonicate,
as well as the supernatant (cytosolic) and pellet (i.e.
membrane) fractions resulting from high speed centrifugation were
subjected to SDS-PAGE, and the proteins were visualized with Coomassie
Blue. Fig. 3 presents the results of
these studies. In the absence of IPTG induction, only a small amount of
27-kDa band was detected (lane 2) and this band was
apparently removed by centrifugation (lane 3). Incubation of
the cells in IPTG resulted in production of substantial amounts of the
27-kDa band (lane 4), but again much of this protein was
pelleted by high-speed centrifugation (lanes 5 and
6). The protein could be mostly solubilized by addition of
0.2% Sarkosyl to the crude homogenates before the centrifugation as
seen in lanes 8 and 9. The solubilized fraction had strong trehalose-P phosphatase activity and could be purified to a
single band as shown below.

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Fig. 3.
SDS-PAGE of TPP fractions isolated from
transfected E. coli. E. coli was
transfected with TPP gene and cells were grown, induced with IPTG
(lanes 4-9), and sonicated as described under
"Results." Lanes 1 and 10, protein standards;
lane 2, crude extract of cells without induction by IPTG;
lane 3, supernatant fraction of extract in lane
2; lanes 4-6, cells induced with IPTG, whole extract,
supernatant fraction after high speed centrifugation, pellet from high
speed centrifugation; lanes 7-9, whole extract, from cells
induced with IPTG, mixed with 0.2% Sarkosyl, supernatant fraction
after high speed centrifugation, and pellet from centrifugation.
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The solubilized protein was applied to a nickel ion column and
after thorough washing in 10 mM imidazole, the column was
eluted batchwise with various concentrations of imidazole. Fig.
4 shows that 250 mM imidazole
eluted a single protein that migrated slightly faster than the 31-kDa
protein standard and this fraction had strong TPP activity. This
protein fraction was used in all subsequent studies to compare its
properties with those of the native TPP isolated from mycobacteria.

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Fig. 4.
Purification of recombinant TPP on the
nickel-chelate column. Lanes 1 and 7,
protein standards; lane 2, crude E. coli
supernatant fraction before application to column; lane 3,
fraction passing through nickel column; lane 4, fraction
eluting with 10 mM imidazole; lane 5, fraction
eluting with 25 mM imidazole; lane 6, fraction
eluting with 250 mM imidazole.
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The expressed protein containing a (His)6 tag was purified
to near homogeneity on a metal ion column. As shown in Fig. 4
(lane 6), the elution from this column showed a single
protein on SDS gels with a molecular weight of about 27,000. As
demonstrated below, this protein had strong phosphatase activity, and
this activity was very specific for trehalose-P.
Properties of the Trehalose-P Phosphatase--
The enzyme purified
from E. coli was characterized in terms of pH optima,
substrate specificity, and metal ion requirements, and other properties
as discussed below. The TPP purified from M. smegmatis and
the recombinant TPP had almost identical properties except for their
requirement for divalent cations (see below).
The pH optimum of TPP was determined using two different buffers as
shown in Fig. 5. The pH optimum of this
activity was 7.0-7.5 using either Tris-HCl or Tris maleate buffers.
The enzyme had almost no activity at either pH 5 or 9, in contrast to
many nonspecific acid or alkaline phosphatases that have maximum
activity at around pH 5.0 or 9.0.

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Fig. 5.
Effect of pH of incubation mixture on
activity of native and recombinant TPP. Incubations were prepared
as described in the text but contained buffers at various pH values as
shown in the figure. Enzyme activity was determined as described under
"Experimental Procedures." Diamonds are results with TPP
purified from Mycobacterium smegmatis. Squares are
results with recombinant enzyme made in E. coli.
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TPP was incubated with a variety of sugar phosphates, tested at 2 and 5 mM concentrations, to determine whether any of them could
serve as substrates for this enzyme. Fig.
6 demonstrates that this enzyme had very
strong phosphatase activity toward trehalose-P, but essentially no
activity with any of the other sugar phosphates. The slight activity
seen with glucose-6-P or glucose-1-P is probably because of small
amounts of inorganic phosphate present in these substrates as a result
of some degradation (i.e. loss of phosphate) during storage,
because the substrate itself gives some background color in the
phosphate determination. The enzyme also had no activity on the general
phosphatase substrate, para-nitrophenyl phosphate. This data
indicates that TPP is an unusual and very specific phosphatase, because
these enzymes usually have a rather broad substrate specificity and can
hydrolyze many different phosphate esters.

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Fig. 6.
Substrate specificity of the native and
recombinant TPPs. Incubations were as described in the text but
contained various sugar-Ps instead of trehalose-P to determine the
specificity of the native M. smegmatis or recombinant
E. coli TPP. With the enzyme from M. smegmatis,
each sugar-P was tested at 2 and 5 mM concentrations,
whereas with the recombinant TPP from E. coli, the sugar
phosphates were tested at 2 mM concentrations.
In each case, activity was determined by the release of phosphate as
described under "Experimental Procedures."
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The effect of trehalose-P concentration on the reaction rate was
determined. The rate of the reaction was proportional to the amount of
trehalose-P in the incubation up to about 5 mM, and the
Km for trehalose-P was calculated to be about 1.6 mM (data not shown).
Native TPP showed an almost absolute requirement for the divalent
cation Mg2+, as shown in Fig.
7. The data in this figure also show that
Mn2+ worked to some extent, but was much less effective
than Mg2+. The optimum concentration of Mg2+
for the native TPP was about 1-2 mM. On the other hand,
the recombinant TPP was somewhat different in regard to its reactivity
with Mn2+ as also shown in Fig. 7, because this TPP showed
almost equal activity with both Mn2+ and Mg2+.
In either case, the optimum concentration of cation was about 3-4
mM. The significance of this difference in cation
requirement between the native and recombinant TPPs is not clear at
this time, but it could be related to the presence of the His tag on
the recombinant protein.

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Fig. 7.
Effect of Mg2+ and/or
Mn2+ concentration on TPP activity. Incubations were
as described in the text but contained various amounts of
Mg2+ or Mn2+ as indicated in the figure. TPP
activity was measured as described under "Experimental Procedures."
Solid lines are experiments using the M. smegmatis native TPP; dashed lines are using the
recombinant TPP from E. coli. Squares are
incubations containing various amounts of Mn2+;
circles are incubations containing Mg2+ as the
cation, rather than Mn2+.
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TPP was subjected to far-UV and near-UV circular dichroism measurements
as described under "Experimental Procedures." A summation of the
data is presented in Table II and
indicates that the protein has about 50%
-pleated sheet, and is
quite compact.
Inhibition of Trehalose-P Phosphatase Activity--
The enzymatic
activity was not inhibited by 10 mM sodium fluoride or 10 mM sodium orthovanadate. It was also not inhibited by a
variety of sugars such as trehalose, maltose, glucose-6-P, glucose-1-P,
GlcNAc-1-P, at up to 10 mM concentrations, nor by 10 mM
-glycerophosphate, or acetyl-coenzyme A. The enzyme
also did not show any phosphatase activity toward any of these
phosphorylated compounds.
Both the natural TPP and the recombinant enzyme were inhibited by two
antibiotics, diumycin and moenomycin as shown in Fig. 8. It can be seen that the activity was
progressively inhibited by adding increasing amounts of either of these
antibiotics to the incubation mixtures, and 50% inhibition by either
antibiotic occurred at about 100 µg/ml. These two antibiotics were
previously shown to inhibit the growth of M. smegmatis
(18).

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Fig. 8.
Inhibition of TPP activity by
moenomycin and diumycin. Incubations were as
described under "Experimental Procedures" except that various
amounts of moenomycin (squares) or diumycin
(circles) were added as shown in the figure. Inhibitors were
added after the addition of substrates and buffers and reactions were
initiated by addition of enzyme. Both native enzyme (solid
lines) and recombinant TPP (dashed lines) were tested
and both were inhibited to the same extent.
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Stability of the Trehalose-P Phosphatase--
The purified enzyme
(both native and recombinant) was stable to freezing and maintained its
activity for at least several weeks when stored at
20 °C. The
enzyme was also quite stable to heating as shown in Fig.
9. Thus, when the enzyme was placed in a
hot water bath at various temperatures from 40 to 60 °C, there was
little loss of activity for at least 6 min of heating. In fact, even at
70 °C, the enzyme retained significant activity for several minutes
and then slowly lost activity. During the purification, we found that
the crude extracts could be heated at 95 °C for 1 or 2 min, and in
many cases most of the TPP activity was retained. However, this
procedure was not completely reproducible from one extract to another,
and therefore the heat step was used at 60 °C.

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Fig. 9.
Heat stability of recombinant TPP. TPP
purified from the metal ion column was incubated for various times at
temperatures ranging from 40 to 100 °C and samples were withdrawn at
the times shown in the figure. Each sample was then tested for its
ability to release inorganic phosphate from trehalose-P. Numbers
above (or below) the lines indicate the
temperature of incubation for each treatment.
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Because the recombinant TPP was found mostly in the membrane fraction
and required treatment with Sarkosyl for solubilization, we tested the
effects of various detergents on enzyme activity. Fig.
10 presents the results obtained with a
variety of detergents at increasing concentrations. It can be seen that
the enzyme activity was rapidly lost in the presence of increasing
concentrations of Sarkosyl or deoxycholate, and at 0.05% of these
detergents there was almost complete loss of activity. On the other
hand, detergents such as Nonidet P-40, Triton X-100, and octyl
glucoside were relatively innocuous and even at 0.2-0.3% there was no
loss of activity. We did use 0.2% Sarkosyl to solubilize the TPP in crude sonicates of the transfected E. coli cells, but the
Sarkosyl was not in the buffer used to elute the TPP from the metal ion columns. This indicates that removal of that detergent results in
restoration of activity.

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Fig. 10.
Effect of various detergents on the activity
of recombinant TPP. Incubation mixtures for the assay of
trehalose-P phosphatase activity contained the components described
under "Experimental Procedures" but also contained various
concentrations of the detergents shown in the figure. Detergents used
were as follows: octyl glucoside (closed triangles), Triton
X-100 (open diamonds); Nonidet P-40 (×-×); deoxycholate
(closed diamonds); Sarkosyl (closed squares). All
of the components including the detergents were added to incubation
tubes and reactions were started by the addition of enzyme. The
formation of inorganic phosphate was measured with malachite green as
described.
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DISCUSSION |
The gene for trehalose-P phosphatase from E. coli
(otsB) has been mapped and located on the E. coli
chromosome (13), and that gene was also identified in the
Arabidopsis thaliana genome by functional complementation of
the yeast trehalose-P phosphatase (tps2) gene (19). In
E. coli, the gene for trehalose-P phosphatase (otsB) and the gene for trehalose-P synthase
(otsA) constitute an operon in which otsB are
proximal to the promoter, and otsA is distal (20). In
Saccharomyces cerevisiae, the synthase and the phosphatase
copurify as a complex and the individual enzymes have not been
separated (21). This complex also contains several regulatory proteins
that affect the activities of these two enzymes. In E. coli,
TPS and TPP apparently do not exist as a complex because the two
proteins could be expressed separately in an otsAB deletion mutant (14). Despite the considerable amount of information on the
genetics of the trehalose pathway in these organisms, very little is
known about the structure of the protein that removes the phosphate
from trehalose-P or the protein that synthesizes trehalose-P (TPS) in
these organisms, nor is there much information on the specificity or
other properties of these two enzymes.
In this report, we describe the purification of the trehalose-P
phosphatase (TPP) from M. smegmatis to near homogeneity and the identification of the complete amino acid sequence of this interesting protein. The translation start of the M. smegmatis TPP is unidentified. The purified polypeptide located
closest to the predicted amino terminus starts at residue 11 (11ALTAVAATPHLLVTSDFDGT30), suggesting that the
actual translation start was located between residues 1 and 11. CLUSTALW alignment of the M. smegmatis TPP predicted amino
acid sequence with those of C. glutamicum and E. coli suggests that the most likely start codon was located at
residue 8 (see Fig. 3). Almost 50% of the amino acids in the 27-kDa
TPP are hydrophobic (i.e., 45 alanines, 7 Phe, 18 Gly, 7 Ile, 28 Leu, and 23 Val), but these amino acids are distributed throughout the protein sequence rather than occurring in specific domains. The hydrophobic nature of the protein may account for some of
its unusual properties. For example, when E. coli cells transfected with the TPP gene were broken by sonication and the crude
homogenate subjected to high speed centrifugation, most of the TPP
activity was found in the pellet, i.e. membrane fraction. However, when 0.2% Sarkosyl was added to the crude homogenate, almost
all of the TPP activity remained in the supernatant fraction. This
protein is also quite resistant to heat denaturation. Thus, the protein
could be heated at temperatures of up to 60 °C for 5 or 6 min with
only a small loss in activity. This property suggests that the protein
is tightly folded and fairly resistant to denaturation. Examination of
the protein by circular dichroism also supports this hypothesis and
indicates that the protein structure is largely in the
-pleated
sheet and is very compact.
Both TPP and TPS are inhibited by the glycoside antibiotics,
moenomycin and diumycin. These antibiotics also inhibit the
growth of M. smegmatis (18). However, because these
compounds also inhibit peptidoglycan synthesis in some organisms, we
cannot be certain that the inhibition of growth of mycobacteria is
because of inhibition in the trehalose biosynthetic pathway. We are
currently designing experiments to determine whether growth inhibition
and inhibition of trehalose synthesis are directly related. We are also
designing experiments to determine whether this trehalose biosynthetic
pathway is essential to the viability of these organisms.