This trehalase shows an almost absolute requirement for inorganic phosphate and Mg2+ for activity see below, and therefore it seemed possible that the enzyme might actually be a phosphor
Trang 1smegmatis ) purification, properties, requirements
J David Carroll1, Irena Pastuszak2, Vineetha K Edavana2, Yuan T Pan2and Alan D Elbein2
1 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Trehalose, i.e a-d-glucopyranosyl-a-d-glucopyranoside,
is a nonreducing disaccharide that is widely distributed
throughout the biological kingdom, being prominent in
prokaryotes and lower eukaryotes, but absent from
mammals [1] It has a number of important and
differ-ent functions in these various organisms, including:
acting as a reservoir of glucose and⁄ or energy [2]; ser-ving as a protectant of proteins and membranes during various stress conditions [3,4]; having a regulatory role
in the control of glucose metabolism [5]; playing a role
in transcriptional regulation [6]; and serving as an essential component of various cell wall glycolipids,
Keywords
effect of phosphate; glycosyl hydrolase;
pyrophosphate inhibition; trehalase;
trehalase inhibitors
Correspondence
A D Elbein, Department of Biochemistry
and Molecular Biology, University of
Arkansas for Medical Sciences, Little Rock,
AR 72205, USA
Fax: +1 501 686 5189
Tel: +1 501 686 5176
E-mail: elbeinaland@uams.edu
(Received 11 October 2006, revised 18
January 2007, accepted 22 January 2007)
doi:10.1111/j.1742-4658.2007.05715.x
Trehalose is a nonreducing disaccharide of glucose (a,a-1,1-glucosyl-glu-cose) that is essential for growth and survival of mycobacteria These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow Mycobacterium smegmatis and
Mycobacteri-um tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose In this study, we report on the purifica-tion and characterizapurifica-tion of the trehalase from M smegmatis, and its com-parison to the trehalase from M tuberculosis Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M smegmatis also requires Mg2+ for activity, whereas the
M tuberculosis trehalase does not require Mg2+ The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evi-dence for a phosphorolytic cleavage, or for any phosphorylated intermedi-ates in the reaction However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function
of the phosphate may involve stabilizing the protein conformation and⁄ or initiating protein aggregation Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits The trehalase is highly specific for a,a-trehalose and did not hydrolyze a,b-trelalose or b,b-trehalose, trehalose dimycolate, or any other a-glucoside or b-glucos-ide Attempts to obtain a trehalase-negative mutant of M smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants This suggests that trehalase is an essential enzyme for these organisms The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg2+ are present Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate
Trang 2especially in mycobacteria and other related bacteria [7].
In some of these organisms, notably mycobacteria and
corynebacteria, there are three different pathways that
can produce trehalose [8,9], and mutants defective in all
three pathways are unable to grow unless the growth
medium contains or is supplemented with trehalose
[10,11] Thus, trehalose is essential for mycobacteria
and corynebacteria
The major enzyme involved in the turnover of
treha-lose, or its conversion to two molecules of glucose, is
trehalase (a,a,1,1-glucosyl hydrolase) [12] Trehalases
(EC 3.2.1.28) are generally placed in glycoside
hydrol-ase family 65 [13], although Mycobacterium smegmatis
MSMEG4528 and Mycobacterium tuberculosis MT2474
and Rv2402 have been placed in glycoside hydrolase
family 15 This group of enzymes is widely distributed
in the biological world, and trehalases are found in
most organisms that synthesize and⁄ or utilize trehalose
In some organisms, such as Saccharomyces cerevisiae,
there are several different trehalases, one of which is
regulated by cAMP and phosphorylation, and another
which is apparently not a regulatory enzyme [14] On
the other hand, in most bacteria, trehalase does not
appear to undergo post-translational modifications
such as phosphorylation [15], although some of these
enzymes may be transcriptionally regulated
The trehalase described in this article was purified to
apparent homogeneity from cytoplasmic extracts of
M smegmatis This enzyme is unusual among this
group of glycosyl hydrolases [13] in that it has an
almost absolute requirement for inorganic phosphate
and Mg2+ for activity, although we cannot find any
evidence for a phosphorylated sugar intermediate in
the reaction The role of the inorganic phosphate and
Mg2+ may be to stabilize the enzyme conformation,
or to aid in aggregation of the protein to produce
act-ive enzyme The trehalase may be an essential enzyme
for mycobacteria, as all attempts to isolate mutants
deleted in this protein were unsuccessful The
proper-ties, amino acid sequence and requirements of this
trehalase are herein described
Results
Purification of M smegmatis trehalase
Trehalase was purified about 160-fold from the
cytoso-lic extract of M smegmatis using the procedures
out-lined in Table 1 The steps in the purification included
ion exchange chromatography on DE-52, hydrophobic
chromatography on phenyl-Sepharose, gel filtration on
Sephacryl S-300, and chromatofocusing With these
procedures, the trehalase was purified about 160-fold,
with an overall yield of about 0.4% Figure 1 shows the protein profiles at each stage of purification, as demonstrated by SDS⁄ PAGE It can be seen in lane 6 that after purification by chromatofocusing, there was one major band with a molecular mass of about
71 kDa on the SDS gels (Fig 1) On the other hand, active trehalase, subjected to gel filtration on Sephacryl S-300, was eluted from the column in the void volume, indicating a molecular mass of over 1.5· 106Da, and suggesting that the active enzyme is a multimer of 20
Table 1 Purification of trehalase from M smegmatis.
Purification steps
Protein (mg)
Activity (units a )
Specific activity (unitsÆmg)1)
Fold of purification
2 (NH)2SO4fraction 2329 636 512 273 1.2
3 DE-52, Phenyl-sepharose
5 Chromatofocusing 0.08 2959 36 992 162
a Units are expressed as nanomoles of glucose released from tre-halose in 1 min at 37 C.
Fig 1 Protein profiles by SDS ⁄ PAGE at various stages in the purifi-cation of M smegmatis trehalase Trehalase was purified as des-cribed in Table 1, and an aliquot of the enzyme preparation at each step in the purification was subjected to SDS ⁄ PAGE for 6 h at
30 mA Proteins were detected by staining with Coomassie Blue Lanes: 1, crude extract; 2, ammonium sulfate fractionation; 3, DE-52 fraction; 4, phenyl-Sepharose fraction; 5, Sephacryl S-300 fraction; 6, preparation after chromatofocusing (the trehalase band
in lane 6 is indicated by arrows); 7, protein standards with masses
of 97 (top band), 66, 45 and 31 kDa.
Trang 3or more subunits The purified enzyme was stable to
storage at ) 20 C for at least several weeks, but lost
activity upon repeated freezing and thawing It could
be stored on ice for several weeks with no apparent
loss of activity
The 71 kDa protein band from lane 6 of the SDS
gels was excised from the gels and subjected to trypsin
digestion and amino acid analysis, with MS being used
to determine the amino acid composition of the various
peptides The resulting peptide sequences were used to
screen the M smegmatis mc2155 genome sequence
maintained by the Institute for Genomic Research
(TIGR)
(http://cmr.tigr.org/tigr-scripts/CMR/Genome-Page.cgi?org_search¼ & org ¼ gms.) Using the
pro-gram tblastn, which compares an amino acid query
sequence against a nucleotide sequence database
dynamically translated in all six reading frames, all of
the trehalase-derived peptide sequences aligned with a
single M smegmatis ORF, MSMEG 4528 This ORF
specifies a 672-residue polypeptide with a calculated
molecular mass of 75.2 kDa tblastn screening of the
M tuberculosis H37Rv genome sequence (http://www
sanger.ac.uk/Projects/M_tuberculosis/) with the amino
acid sequence predicted by MSMEG 4528 identified a
homologous M tuberculosis ORF, Rv2402 Rv2402
specifies a 642-residue protein, annotated as a
‘con-served hypothetical protein’ of unknown function The
respective predicted amino acid sequences of
MSMEG 4528 and Rv2402 are 88% identical, with the
identity distributed evenly throughout the sequence
alignment The comparison of these two sequences is
presented in Fig 2 The sequence alignment of the
M smegmatis trehalase also showed 72% identity
with a hypothetical trehalase from Nocardia farcinica,
63% identity with a proposed trehalase from Frankia,
37% identity with that protein in Burkholderia mallei,
31% identity with Corynebacterium efficiens, 24%
iden-tity with Aspergillus fumigatus, and 28% ideniden-tity with
Schizosaccharomyces pombe
Cloning and expression of M smegmatis
trehalase
The 2019 bp MSMEG 4528 ORF was PCR amplified
from M smegmatis mc2155 genomic DNA using the
oligonucleotide primers pET100 TOPO StreFP 5¢-CA
CC ATG ATG TGC TGC ATG GTT CTG CAA CA
GA-3¢ and pET100 TOPO treFP 5¢-TGA GCG TCA
CAT CGG GGC GTT-3¢ The pET100 TOPO StreFP
includes the 4 bp sequence ‘CACC’ (underlined
nucleo-tides) necessary for directional cloning on the 5¢-end
The bold ‘ATG’ in the FP represents the start codon
and the bold ‘TCA’ in pET100 TOPO StreFP represents
the stop codon of the recombinant ORF The PCR product was amplified and ligated with the precut pET100D-TOPO (Invitrogen), generating the plasmid pSTRE TOPO The entire cloned (His)6–Stre gene fusion was sequenced to confirm the fidelity of the amplification pSTRE TOPO was transformed into the Escherichia coli expression strain BL21 star (DE3) pSTRE TOPO in BL21 star (DE3) was used for further expression studies
Properties of the trehalase purified from
M smegmatis Effect of time and protein concentration on the activity and characterization of the product
The conversion of trehalose to glucose was measured
by determining the amount of reducing sugar resulting from the hydrolysis of trehalose The formation of reducing sugar increased in a linear fashion with increasing time of incubation for at least 1 h, and was also linear with the amount of enzyme added up to
100 lg of protein (data not shown) These data estab-lished that all measurements were being made in the linear range of measurements
Glucose was the only product identified, both at early times of incubation and with longer incubations Glucose was identified by paper chromatography in several different solvents that readily separate this sugar from other monosaccharides, such as mannose and galactose, and other disaccharides such as maltose, trehalose and cellobiose It was also identified by HPLC on the Dionex Carbohydrate Analyzer, which also readily separates the various monosaccharides The resulting d-glucose was also determined using the glucose oxidase reagent kit, which is specific for d-glu-cose Measurements using glucose oxidase to determine the amount of glucose released gave very similar values
to those obtained using the reducing sugar test to measure the amount of glucose
This trehalase shows an almost absolute requirement for inorganic phosphate and Mg2+ for activity (see below), and therefore it seemed possible that the enzyme might actually be a phosphorylase, rather than
a glucosyl hydrolase Therefore, a variety of experi-ments were done to determine whether any phosphor-ylated intermediates were produced in this reaction Thus, the above assay mixtures were removed after various times of incubation and were carefully ana-lyzed for the presence of glucose 1-phosphate or glucose 6-phosphate To do this, incubation mixtures were passed through columns of DE-52 or
Dowex-1-Cl– to bind any possible phosphorylated sugars, and the columns were then eluted with ammonium
Trang 4Fig 2 CLUSTALW alignment of predicted amino acid sequences of M smegmatis trehalase MSMEG 4528 and putative M tuberculosis Rv2402 Numbering refers to the individual sequences, rather than to the alignment Conserved residues and strong and weak conservative substitutions are indicated by ‘*’, ‘:’ and ‘.’, respectively Gaps introduced by CLUSTAL to optimize the alignment are indicated by ‘–’ Polypep-tide fragments used to identify the M smegmatis ORF are underlined.
Trang 5bicarbonate to remove such phosphorylated sugars.
These eluates were analyzed for the presence of sugar
phosphates No evidence for the presence of glucose
phosphate could be obtained either in short-time
incu-bations, or in longer incubations
As phosphorylases can also be assayed in the
direc-tion of synthesis, various incubadirec-tions were also
pre-pared using either a-glucose 1-phosphate or b-glucose
1-phosphate plus free glucose In this case, the assay
was set up to measure the formation of trehalose All
of these assays were also negative for trehalose
forma-tion
Requirements for enzyme activity
The purified trehalase demonstrated a requirement for
inorganic phosphate, which could also serve as the
buffer in the reaction Therefore, the effects of a
vari-ety of buffers, all tested at 100 mm and pH 7.0, on the
trehalase activity were determined, in the presence and
absence of added sodium (or potassium) phosphate,
and also in the presence of sodium arsenate rather
than phosphate The results of these experiments are
presented in Table 2 It can be seen that in the absence
of added potassium (or sodium) phosphate, none of
the other buffers (Hepes, Tris, acetate, citrate, borate,
Mops or Mes) were able to activate the trehalase, as compared to control incubations with only phosphate buffer at pH 7.0 However, when 100 mm phosphate was added to any of these incubations, all of them (except for those incubations containing citrate or Mes buffer) gave the same amount of trehalase activity as the incubation with phosphate buffer alone The inab-ility of citrate to act as a favorable buffer may be due
to its strong chelation activity, as it probably competes favorably with the trehalase for the Mg2+also needed for stimulation Interestingly, arsenate was able to sub-stitute for phosphate to some extent with some of these buffers, but it could not replace phosphate when either acetate, borate or Tris were used as the buffer The studies described here were done with M smegma-tisB11 Also shown in Table 2 and discussed in a later section are comparative results with the trehalase parti-ally purified from M tuberculosis H37Rv
As indicated above, the mycobacterial trehalase showed an absolute requirement for inorganic phos-phate (Fig 3), with optimum activity being observed at
a concentration of 100 mm Although phosphate could also serve as the buffer for the reaction, the require-ment for phosphate was independent of the buffer used,
as indicated in Table 2 Figure 3 demonstrates that
Table 2 Effects of various buffers on the activity of trehalase.
Buffer
Trehalase activity (A 540 nm )
M smegmatis B11
M tuberculosis H37Rv
Fig 3 Effect of inorganic phosphate and arsenate on the activity of the mycobacterial trehalase Incubations contained 100 m M Hepes buffer (pH 7.1), 50 m M trehalose, 6 m M MgCl2, various amounts of sodium phosphate (r – r) or sodium arsenate (m – m) and 15 units of purified trehalase, all in a final volume of 100 lL After an incubation of 6 min at 37 C, reactions were stopped by heating in
a boiling water bath for 5 min, and the amount of glucose produced was determined by the reducing sugar test, or by the glucose oxid-ase assay method One unit is defined as that amount of trehaloxid-ase that produces 1 nmol of glucose in 1 min at 37 C.
Trang 6arsenate could substitute for the phosphate requirement
to some extent, although it was not as effective as
phate However, it did give the same profile as
phos-phate at various arsenate concentrations, suggesting
that it had the same effect on the enzyme The only
product produced from trehalose in the presence of
arsenate was also identified as glucose by the glucose
oxidase reaction and by HPLC on the Dionex
Carbo-hydrate Analyzer Although arsenate was somewhat
effective in replacing phosphate as the activator of
treh-alase, in the presence of 100 mm phosphate increasing
concentrations of arsenate inhibited the reaction, and
at equal concentrations of phosphate and arsenate
(100 mm each), the production of glucose was inhibited
by 40%
The enzyme also showed an absolute requirement
for Mg2+, with optimum activity occurring at
concen-trations of 3.5–4 mm (Fig 4) Mg2+ could not be
replaced by Ca2+, Mn2+ or Zn2+ (data not shown)
The pH optimum for trehalase when potassium phos-phate was used as the buffer, at 6 mm MgCl2, was found to be 7.1 (data not shown) The trehalase activ-ity dropped sharply at pH values of 6.5 and below, as well as at pH values of 8.0 and above Many glycosyl hydrolases, including a number of trehalases, have pH optima at about 5.0–5.5 In addition, these enzymes generally do not show any requirements for inorganic phosphate, and most are not activated by metal ions (Table 3)
Substrate specificity and concentration for optimum activity
Several glucose disaccharides were tested as possible substrates for the purified trehalase, including maltose (4-O-a-d-glucopyranosyl-d-glucopyranoside), isomal-tose (6-O-a-d-glucopyranosyl-d-glucopyranoside), sucrose (O-b-d-fructofuranosyl-(2fi 1)-a-d-glucopyranoside), cellobiose (4-O-b-d-glucopyranosyl-d-glucopyranoside), p-nitrophenyl-a-d-glucopyranoside, and methyl-a-d-glucopyranoside None of these compounds was hydro-lyzed by the trehalase, even when added to incubation mixtures at high concentrations (50 mm) (data not shown) The trehalase also did not hydrolyze a,b-treha-lose or b,b-trehalose, a,a-trehalose-6,6¢-dibehenate, trehalulose or nigerose (3-O-a-d-glucopyranosyl-d-glucopyranoside) Trehalase also was inactive on treha-lose dimycolate, an important glycolipid found in the cell wall of M tuberculosis, and other mycobacteria [7]
In these cases, assays were done by determining the release of free glucose with the glucose oxidase reagent kit, and by HPLC on the Dionex Carbohydrate Ana-lyzer The enzyme also did not show any activity with glucose 1-phosphate, glucose 6-phosphate, mannose 1-phosphate or trehalose 6-phosphate, either by glucose oxidase assay or by HPLC (data not shown)
Several of the above sugars were also tested for their ability to inhibit the activity of trehalase on a,a-trehalose Only methyl-a-d-glucopyranoside showed any inhibitory effect, giving about 50% inhibi-tion at a concentrainhibi-tion of about 12 mm
0
.
0 2
.
0 4
.
0 6
.
0 8
Cati n c n c entrati n ( m M )
Fig 4 Effect of Mg 2+ concentration on the activity of the M
smeg-matis trehalase Reaction mixtures were as described in the text,
with 100 m M sodium phosphate buffer (pH 7.1), 50 m M trehalose,
5 units of purified trehalase, and increasing amounts of MgCl2as
indicated After an incubation of 15 min, reactions were stopped by
heating, and the amount of glucose formed was determined as
indi-cated in Fig 3.
Table 3 Comparison of trehalases from various organisms.
Trang 7The enzymatic activity increased with increasing
concentrations of a,a-trehalose in the incubations up
to about 50 mm (Fig 5) A plot of this data by the
method of Lineweaver and Burk is shown in the inset,
and this plot indicated that the Km for trehalose was
about 20 mm
Table 3 compares some of the properties of the
M smegmatis trehalase to those of some of the other
trehalases that have been isolated and purified from
various other organisms, and at least partially
charac-terized Most of these enzymes are from fungi or yeast,
and have molecular masses of about 100–150 kDa, as
compared to the mycobacterial enzyme, which appears
to be a multimer of 20 or so identical subunits These
trehalases also vary in terms of pH optima, from
aci-dic trehalases with optima at 4.0–5.6 to neutral
treha-lases with pH optima of about 7, like the mycobacterial enzyme In a few cases, such as the yeast neutral trehalase and the mycobacterial trehalase, the Km for trehalose is quite high (34 and 20 mm, respectively), but many of the Kmvalues are in the low millimolar range Finally, only two of the trehalases have a requirement for a divalent metal ion The Fusa-rium oxysporumneutral trehalase requires Ca2+, which
is apparently involved in stabilizing the protein, and the M smegmatis trehalase requires Mg2+, which appears to be necessary for activity However, only the mycobacterial trehalases seem to also require phos-phate in order to be active
Effect of various phosphorylated compounds
on trehalase activity Trehalase activity was inhibited by pyrophosphate and polyphosphates Figure 6 shows the effects of increas-ing concentrations of potassium pyrophosphate on the hydrolysis of trehalose (formation of glucose) at var-ious concentrations of phosphate buffer Thus, with
1 mm pyrophosphate, there was little or no effect on trehalase activity, even at lower concentrations of phosphate buffer However, it can be seen that at a
A
B
Fig 5 Effect of substrate concentration on trehalase activity
Var-ious amounts of trehalose were added to incubation mixtures
con-taining 100 m M sodium phosphate buffer (pH 7.1), 6 m M MgCl 2 ,
and 15 units of purified trehalase After an incubation of 10 min at
37 C, the amount of glucose produced was determined (A) These
data were plotted by the method of Lineweaver and Burke, as
shown in (B).
Fig 6 Inhibition of trehalase activity by sodium pyrophosphate Incubation mixtures contained 100 m M sodium phosphate buffer (pH 7.1), 6 m M MgCl 2 , 50 m M trehalose, 15 units of purified treh-alase, and various amounts of sodium pyrophosphate as follows: (r – r), no pyrophosphate; (h – h), 1 m M sodium pyrophosphate; (m – m), 4 m M sodium pyrophosphate; ( · — · ), 8 m M sodium pyrophosphate; (* – *), 16 m M sodium pyrophosphate After an incubation of 10 min, reactions were stopped by heating, and the amount of glucose produced was measured.
Trang 8pyrophosphate concentration of 8 mm, trehalase was
inhibited by more than 50%, even at phosphate
con-centrations of 100 mm At 16 mm pyrophosphate,
there was almost complete inhibition of the enzyme,
even at concentrations of phosphate that were 10-fold
higher
Polyphosphates were also inhibitors of the trehalase,
and these compounds were better inhibitors than
pyro-phosphate Four differently sized polyphosphates were
tested, varying in polymerization number from 8 to 60
All of these polyphosphates were quite similar in
inhib-itory activity, causing 50% inhibition of trehalase
activity when added to incubations at about 100 lg
(approximately 150 nmol for a polymerization number
of 8) These incubations also contained 100 mm
potas-sium phosphate buffer Other phosphorylated
com-pounds were also inhibitory to varying degrees ATP
caused about 90% inhibition at a concentration of
20 mm, whereas this concentration of AMP caused
about 50% inhibition UTP also caused about 90%
inhibition at a concentration of 10 mm Sodium
ortho-vanadate, an inhibitor of phosphatases, also inhibited
the trehalase by 80% at a concentration of 10 mm, but
no inhibition was observed with sodium fluoride, even
at 40 mm
Why does the trehalase require inorganic phosphate?
As shown above, the M smegmatis B11 trehalase
requires inorganic phosphate and Mg2+ in order to
catalyze trehalose hydrolysis However, no evidence
for phosphorolytic activity could be demonstrated,
suggesting that phosphate is not directly involved in
the catalytic mechanism Thus, the question arose as
to whether phosphate might affect the conformation
of the enzyme In order to determine whether
inor-ganic phosphate bound to the enzyme, [32P]inorganic
phosphate was incubated with the purified trehalase
in the presence of Mg2+, but without trehalose, for
several minutes on ice, and the mixture was applied
to a Sephacryl S-300 column in the cold The column
was eluted with buffer, and fractions of 2 mL were
collected and assayed for trehalase activity and for
radioactivity As seen in Fig 7, the trehalase emerged
in the void volume (fraction 31¼ 62 mL) of a
Seph-acryl S-300 column, and a peak of [32P]inorganic
phosphate emerged in the same area of the column
and with the same profile, suggesting that inorganic
phosphate was binding to the protein Although this
experiment still does not identify a role for inorganic
phosphate in the mechanism of trehalase activity, it
suggests that phosphate may be involved in causing
or expediting the correct conformation of the
treh-alase, or in causing the aggregation of the trehalase
into its active form
Stabilization of the trehalase by phosphate and magnesium
One of the possible roles for phosphate in the activity
of the trehalase is to stabilize the enzyme and maintain
it in the stable and perhaps aggregated state In order
to determine whether phosphate plays a role in stabil-ity, trehalase was incubated at 50C for various times
in sodium phosphate buffer and⁄ or Mg2+, plus either trehalose or polyethyleneglycol, and the activity of the enzyme was determined at different times of incuba-tion The results of this experiment are shown in Fig 8 It can be seen from the upper profile that opti-mal stability, i.e very little loss of trehalase activity at
50C for 30 min, occurred in the presence of 100 mm phosphate buffer containing 6 mm MgCl2 plus 10% polyethylene glycol Omitting the polyethylene glycol
or replacing it with NaCl, but still heating the enzyme
in the presence of phosphate buffer and Mg2+, resul-ted in reasonable stability, but significantly less than obtained with the above incubation with the polyethy-lene glycol On the other hand, in the presence of
100 mm phosphate buffer alone or phosphate buffer with 50 mm trehalose (see profiles 1 and 3), the enzyme lost most of its activity within 5 min Incubations with
0
.
0 4
.
0 8
.
1 2
.
1 6
r
F a ion N c t umber
-3 )
b
Fig 7 Binding of inorganic phosphate to the purified trehalase [ 32 P]Inorganic phosphate was mixed with purified trehalase and allowed to incubate for 5 min on ice The mixture was then added
to a 1.6 · 110 cm column of Sephracryl S-300, and the column was eluted with 20 m M Tris ⁄ HCl buffer at pH 7.0 Fractions were collec-ted and assayed for radioactivity (n – n) and for trehalase activity (r–r) Arrows indicate the position of the void volume (arrow a) and the molecular mass standards thyroglobulin (669 kDa, arrow b), b-amylase (200 kDa, arrow c), alcohol dehydrogenase (150 kDa, arrow d) and bovine serum albumin (66 kDa, arrow e).
Trang 9phosphate buffer and polyethylene glycol but without
Mg2+ gave slightly more stability than phosphate
alone, but much less than incubations with Mg2+
included In addition, incubations in 100 mm Hepes
buffer (pH 7.1) or 100 mm Hepes buffer (pH 7.1) plus
6 mm MgCl2 did not provide any additional stability
over that seen in the incubations with the enzyme only
in phosphate buffer These results indicate that both
phosphate buffer and Mg2+ are necessary to stabilize
the trehalase against heat denaturation There is a
pre-cedent for inorganic phosphate affecting the
quarter-nary structure of a protein and causing monomers to
aggregrate into dimers [16]
Inhibitors of the mycobacterial trehalase
Validoxylamine has been reported to be an inhibitor of
various trehalases [17,18] We tested the effects of this
drug on the trehalase purified from M smegmatis Val-idoxylamine also inhibited this trehalase, and as shown
in Fig 9, this inhibition was of a competitive nature The Ki for validoxylamine on the trehalase was calcu-lated to be 5· 10)7m
Another known trehalase inhibitor is trehazolin [18,19] However, trehazolin had no effect on the mycobacterial trehalase, indicating that this phosphate-dependent trehalase is different from other trehalases The mycobacterial enzyme was also inhibited by the a-glucosidase inhibitor castanospermine [20], which caused about 50% inhibition at a concentration of
500 lgÆmL)1
Isolation of recombinant trehalase from E coli The E coli expression strain BL21 was transformed with the plasmid pSTRE TOPO, as described in Experimental procedures The cells were grown in LB medium containing 100 lgÆmL)1ampicillin Incubation
of the cells with isopropyl thio-b-d-galactoside resulted
in the production of substantial amounts of protein, but the expressed trehalase protein was associated with the membrane fraction after centrifugation of the soni-cated cells and was presumably in inclusion bodies This protein could be solubilized by the addition of
Fig 8 Effect of phosphate and Mg2+on the heat stability of the
purified trehalase Trehalase was incubated for various times at
50 C under the following conditions: 1 (h – h), 100 m M sodium
phosphate buffer (pH 7.0); 2 (j – j), 100 m M sodium phosphate
buffer (pH 7.0) + 10% polyethylene glycol; 3 (n – n), 100 m M
sodium phosphate buffer (pH 7.0) + 50 m M trehalose; 4 (m – m),
100 m M sodium phosphate buffer (pH 7.0) + 50 m M
treha-lose + 10% polyethylene glycol; 5 (e – e), 100 m M sodium
phos-phate buffer (pH 7.0) + 6 m M MgCl2; 6 (r – r), 100 m M sodium
phosphate buffer (pH 7.0) + 6 m M MgCl 2 + 10% polyethylene
gly-col; 7 ( · — · ), 100 m M sodium phosphate buffer (pH 7.0) + 6 m M
MgCl2+ 500 m M NaCl; 8 (– –) 100 m M Hepes buffer (pH 7.0); 9
(* – *), 100 m M Hepes buffer (pH 7.0) + 6 m M MgCl 2 An equal
ali-quot of each incubation mixture was removed at the times
indica-ted, and assayed for its ability to catalyze the formation of glucose
from trehalose.
Fig 9 Inhibition of trehalase by validoxylamine Incubation mixtures were prepared as described in the legends to other figures, and contained 100 m M sodium phosphate buffer (pH 7.1), 6 m M MgCl2, and 50 m M trehalose, all in a final volume of 100 lL Various amounts of validoxylamine, from 0 to 100 ng, were then added to these incubations, and the reactions were initiated by adding
10 units of purified trehalase to each assay mixture Tubes were incubated for 15 min at 37 C, and the amount of glucose produced was determined by the reducing sugar test.
Trang 100.5% sarkosyl with 1 mm EDTA to the crude sonicate
before centrifugation The expressed protein containing
a (His)6tag was purified on a nickel column The
elut-ed fraction showelut-ed a single protein with a molecular
mass of about 70 kDa, but we were unable to find any
trehalase activity in the solubilized fraction or in the
purified fraction from the column
Partial characterization of the trehalase from
M tuberculosis
The M tuberculosis trehalase was isolated from
cytoso-lic extracts and partially purified by ammonium sulfate
fractionation and chromatography on a column of
Sephracryl S-300 This partially purified enzyme also
exhibited an absolute requirement for inorganic
phos-phate, as seen in Table 2 However, this enzyme does
not appear to require Mg2+for activity, and as shown
in Table 2, this enzyme showed very good activity in
citrate buffer that contained added inorganic
phos-phate On the other hand, the enzyme purified from
M smegmatis was not active in citrate even when
phosphate was present It seems likely that this
inhibi-tion of activity of the M smegmatis enzyme is due to
the lack of Mg2+, as it is probably chelated by the
cit-ric acid The gene for trehalase in M tuberculosis has
over 85% identity at the amino acid level to the
M smegmatis trehalase gene (Fig 2) Interestingly, the
M tuberculosis trehalase is not active in Tris buffer,
even when 100 mm phosphate is added (Table 2),
whereas the M smegmatis trehalase is active in Tris
buffer with added phosphate This may represent
another difference between these two trehalases Other
trehalases have been reported to be inhibited by Tris
buffer [21]
Confirmation of M smegmatis MSMEG 4528
as a trehalase
The identity of MSMEG 4528 as a trehalase was
confirmed by ligating the ORF into the mycobacterial
acetamide-inducible expression vector pSD24 [22] The
resulting chimeric ORF contained the first codons of the
mycobacterial acetamidase gene amiE fused in frame
with the MSMEG 4528 coding sequence The entire
pami-MSMEG4528 cassette was excised from pSD24
and inserted into the single-copy integrating shuttle
plasmid pMV306, generating the plasmid p996A661
This was electroporated into M smegmatis mc2155, and
kanamycin-resistant colonies were recovered These
transformants contained a stable integrated single
copy of the acetamide-inducible M smegmatis
treh-alase A representative transformant and M smegmatis
containing nonrecombinant pMV306 were cultured with varying concentrations of acetamide as previously des-cribed [22] The cultures were then harvested and assayed for trehalase activity The transformant strain,
M smegmatis p996A661, exhibited an acetamide-dependent increase in trehalase activity as shown in Table 4, whereas the strain containing pMV306 was unaffected by the addition of acetamide
Discussion
The trehalase described in this report is unusual as far
as glycosyl hydrolases are concerned, as it requires inorganic phosphate and Mg2+ for activity
Trehalas-es and other glycosyl hydrolasTrehalas-es in glycoside hydrol-ase family 65 or glycoside family 15 do not have a requirement for inorganic phosphate for activity, unless they are phosphorylases The reason for this requirement by the mycobacterial trehalase is still not known Several experiments were done to determine whether a phosphorylated sugar intermediate was involved in the reaction, and⁄ or whether the product
of the reaction was actually glucose 1-phosphate or glucose 6-phosphate All of these experiments gave negative results However, an experiment in which radioactive inorganic phosphate was incubated briefly with purified trehalase did suggest that the phosphate was bound to the enzyme, as active trehalase itself emerges in the void volume of a Sephracryl S-300 col-umn, and the mixture of radioactive phosphate and purified enzyme also emerged in the void volume area, both being eluted with the same profile This experi-ment, and several experiments showing that the treh-alase was most stable to heating (i.e up to 30 min at
50C) when it was in 100 mm phosphate buffer con-taining 6 mm MgCl2 plus 10% polyethylene glycol, suggest a role for phosphate as a stabilizer, and per-haps as an effector of an aggregated and active treh-alase conformation
Incubation of the trehalase in other buffers in the absence of inorganic phosphate or MgCl2 resulted in substantial loss of activity in 2 min, at 40 C or higher Thus it seems that at least one function of the
phos-Table 4 MSMEG 4528 codes for trehalase activity.
Acetamide (m M )
Trehalase activity (nmol Glc per mg protein)
M smegmatis (p996A661) M smegmatis (pMV306)