(Received for publication, May 2, 1997)
From the Fakultät für Biologie,
§ Fakultät für Chemie, Periplasmic In Escherichia coli, maltodextrins enter the periplasm
preferentially via the outer membrane porin LamB (1). In the periplasm, maltodextrins are either cleaved by a periplasmic amylase, MalS, or are
transported to the cytoplasm via the binding
protein-dependent transport complex MalEFGK2
(2). This transport system can only transport maltodextrins up to
maltoheptaose. Longer dextrins have to be cleaved prior to transport.
In the cytoplasm, linear maltodextrins are processed by maltodextrin
phosphorylase, amylomaltase, or maltodextrin glucosidase (3-5).
The malS gene, located at 80.54 min on the E. coli chromosome, is part of the complex maltose regulon (6), which
is involved in the uptake and degradation of maltodextrins.
Thus, malS is controlled by MalT, the positive regulator of
the system (7).
MalS hydrolyzes In this study we describe an improved purification procedure,
biochemical characterization and mass spectrometric identification of
the disulfide bond structure of native MalS. Mass spectrometric molecular weight analyses using electrospray-ionization mass
spectrometry (ESI-MS)1 and
matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS) peptide mapping analyses were applied for identification of
the molecular structure of native MalS. Proper formation of disulfide
bonds is crucial for attaining the correct three-dimensional structure
of proteins (10-13). Therefore, it was important to determine the
location of all disulfide bonds in MalS. Mass spectrometric methods
have successfully been employed for the study of disulfide bridges of
peptides and proteins (14-18), and several comprehensive reviews are
available on this subject (19-21). In our studies we used proteolytic
cleavage techniques, followed by high performance liquid chromatography
(HPLC) separation and MALDI-MS for the rapid identification of the
disulfide bridges.
E. coli strains are
derivatives of MC4100 which is F Strain CS66 expressing malS
from pUMA103 was grown in minimal medium 9 (M9) (26) supplemented with
0.2% casamino acids as carbon source. Cells from a 2.5-liter overnight
culture containing 100 µg/ml ampicillin were harvested by
centrifugation (10 min, 5000 rpm, 21 °C, GS-3 rotor) and resuspended
in 100 ml of 10 mM Tris-HCl, pH 7.5, prior to preparation
of cold osmotic shock fractions. The cold osmotic shock procedure was
carried out according to Neu and Heppel (27) with the exception of
using CaCl2 instead of MgCl2. Remaining cells
were removed by centrifugation (9000 rpm, 30 min, 4 °C, SS34 rotor).
Subsequently, the periplasmic extract was lyophilized, and proteins
were solubilized in buffer A (20 mM Tris-HCl, pH 7.5) at a
concentration of 10 mg/ml. After dialysis overnight in buffer A,
insoluble proteins were removed by centrifugation (9000 rpm, 15 min,
4 °C, SS34 rotor).
The resulting supernatant fraction was loaded on a MonoQ column (from
Pharmacia Biotech Inc.), which had been equilibrated with buffer A. Non-interacting proteins were washed off with 10 ml of buffer A, and
the remaining proteins were eluted with a linear gradient from 0 to 200 mM NaCl in 40 ml of buffer A at a flow rate of 0.5 ml/min.
MalS eluted at about 80 mM NaCl.
SDS-PAGE was done as described by Laemmli (28). SDS gels were stained
with Coomassie Blue (29). To detect MalS on Western blots, polyclonal
antiserum against MalS was used.
For mass spectrometric structure determination, MalS solutions were
concentrated by ultrafiltration using microconcentrator devices
(Amicon, microcon, cutoff 10 kDa). Retentates were washed with 500 µl
of 50 mM ammonium bicarbonate, pH 8, and collected to
result in a final protein concentration of 6.9 mg/ml. Aliquots were
used for further investigations.
Amylase activity was determined
using p-nitrophenylhexaoside (PNP6) as a substrate. The
release of p-nitrophenol from PNP6 by MalS was assayed at
room temperature.
Amylase assays of whole cells were performed as described with the
exception that cells were grown overnight in M9 medium supplemented
with 0.2% casamino acids and 100 µg/ml ampicillin (23).
Activity of purified MalS was determined in an assay buffer of 50 mM Tris-HCl, pH 8.0, 5 mM CaCl2.
Assays were carried out in a total volume of 125 µl at room
temperature. MalS was added to a final concentration of 1.16 µg/ml
(15.7 nM). The reaction was started by adding PNP6 to a
final concentration of 1 mM. To determine the effect of
various effectors, preincubation of MalS with the corresponding
substance was performed for 5 min at room temperature prior to starting
the reaction. After appearance of a pale yellow color the absorbance at
405 nm was determined with an Anthos HTII microplate reader. For
determination of kinetic parameters, MalS was used at a concentration
of 1.45 µg/ml, and PNP6 concentrations between 0.5 and 500 µM were assayed. All assays were performed at least in
duplicate. Results varied by <10%.
Alkaline phosphatase
activity in whole cells was assayed by measuring the rate of
p-nitrophenyl phosphate hydrolysis by permeabilized cells as
described (30). 1 unit of alkaline phosphatase activity corresponds to
1 µmol of p-nitrophenyl phosphate hydrolysis per min at
room temperature. Specific activity is given as units per mg of
cellular protein.
1.7 µM MalS was
reduced by incubation for 15 min in 30 mM DTT (final
concentration). Reoxidation was obtained by adding oxidized glutathione
to 130 mM. After incubating 40 min at room temperature, iodoacetamide was added to a final concentration of 150 mM,
and after further incubation for 5 min at room temperature samples were
precipitated with methanol/CHCl3 (31). The resultant pellet was dissolved in sample buffer and subjected to SDS-PAGE.
The protein was diluted in 20 mM triethanolamine buffer, pH 7.5, to concentrations from
12.5-50 µg/ml and was adsorbed on hydrophilized carbon films,
briefly fixed in 0.1% glutaraldehyde in phosphate-buffered saline,
washed in water, and negatively stained by 2% uranyl acetate, pH 4.5, or 1% phosphotungstic acid, pH 7.2. Reduced MalS was obtained by
incubation with 30 mM DTT prior to adsorption on carbon
films. In addition, specimens were prepared by the glycerol spray
technique and shadow casted by platinum/carbon as described (32).
Specimens were examined in a EM10 C2 (Zeiss, Oberkochen, Germany)
under 80 kV at primary magnification of 36,000 ×.
Experiments were carried out in a Beckman
Instruments Optima XL-A analytical ultracentrifuge. A solution of
A280 = 0.1-0.2 (corresponding to a
concentration of 0.6-1.1 µM in terms of protein monomer
concentration) in a buffer containing 20 mM Tris-HCl, pH
7.5, and 75 mM NaCl was measured at 10,000 rpm at 4 °C.
For the determination of the molecular weight the data were fit to Equation 1.
Biophysik der Makromoleküle,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amylase of Escherichia
coli, the malS gene product, hydrolyzes linear
maltodextrins. The purified enzyme exhibited a Km
of 49 µM and a Vmax of 0.36 µmol of p-nitrophenylhexaoside hydrolyzed per min per mg
of protein. Amylase activity was optimal at pH 8 and was dependent on
divalent cations such as Ca2+. MalS exhibited altered
migration on SDS-polyacrylamide gel electrophoresis under nonreducing
conditions. Analytical ultracentrifugation and electrospray mass
spectrometry indicated that MalS is monomeric. The four cysteine
residues are involved in intramolecular disulfide bonds. To map
disulfide bonds, MalS was proteolytically digested. The resulting
peptides were separated by reverse phase-high performance liquid
chromatography, and matrix-assisted laser desorption/ionization mass
spectrometry analysis indicated the presence of two disulfide bonds,
i.e. Cys40-58 and Cys104-520. The
disulfide bond at Cys40-58 is located in an N-terminal
extension of about 160 amino acids which has no homology to other
amylases but to the proposed peptide binding domain of GroEL, the Hsp60
of E. coli. The N-terminal extension is linked to the
C-terminal amylase domain via disulfide bond
Cys104-520. Reduction of disulfide bonds by dithiothreitol
treatment led to aggregation suggesting that the N terminus of MalS may
represent an internal chaperone domain.
-(1,4) glycosidic linkages in long maltodextrins but
not in maltose. MalS is an enzyme of 659 amino acids with a molecular
mass of 74 kDa (8). It belongs to the
-amylase family that shares a
characteristic (
/
)8-barrel domain containing the
active site (9). The four best conserved regions that are present in
the active site of the
-amylase family are located toward the C
terminus of MalS, i.e. between amino acids 304 and 566 (9).
From amino acid sequence alignments it can be concluded that MalS has
an N-terminal extension of about 160 amino acids of unknown function
(8, 9). Four Cys residues are found at positions 40, 58, 104, and 520 of the mature MalS protein, three of which are located in the
N-terminal extension of MalS, which is not homologous to other
amylases.
Bacteria and Plasmids
lacU169 araD136
rbsR relA rpsL thi (22). CS10 is MC4100
malTcon
malQ malP-lacZ
malS
malZ
dex7. CS4 is CS10, trxB::kan. CS5 is CS10,
gsh::Tn10kan. CS14 is CS4,
ara714 leu::Tn10. CS16 is CS5,
ara714 leu::Tn10. CS66 is CS10,
malE::Tn10. pUMA103 is a pBR322-derived
plasmid that expresses malS under its own promoter (8). pCS7
is a pBAD18s derivative and expresses malS with a deletion
of its signal sequence under control of the arabinose promoter (23).
pBAD18s is a pBR322-derived high copy number plasmid that has the
arabinose promoter followed by a linker containing multiple restriction
sites (24). pAID135 encodes alkaline phosphatase with a signal sequence
deletion (
2-22) (25).
In Equation 1 Ar and
A0 are the absorbance at a radial position
r and at the meniscus r0 (a reference
position), respectively. The parameter M is the molecular
weight of the macromolecule that is derived from the fit;
(Eq. 1)
is the
partial specific volume that was calculated from the amino acid
composition of the protein to be 0.720 ml g
1 (33, 34);
is the density of the solvent that was measured to be 1.01 g
ml
1;
is the angular velocity; R is the
universal gas constant; T is the absolute temperature, and
E is the base line that was determined at 10,000 rpm after
sedimentation of the protein sample at 48,000 rpm for 6 h.
A MalS solution (50 µl; 2 mg/ml) in 50 mM ammonium bicarbonate, pH 7.5, was mixed with a solution (2.7 µl) of Tris-(2-carboxyethyl)phosphin (TCEP) in 10 mM ammonium bicarbonate, pH 7.5 (35, 36). Final concentration of TCEP was 5.4 mM, resulting in a molar ratio of MalS to TCEP of 1:200 (4 Cys per MalS). The solution was incubated at 37 °C for 1 h. A 100 mM iodoacetamide solution in H2O (0.7 µl) was added, and the mixture was incubated at 23 °C for 30 min in the dark, pH 7.5. The molar ratio of MalS to iodoacetamide was 1:50. The reaction was terminated by ultrafiltration using a microconcentrator device (Amicon, microcon, cutoff 10 kDa). Retentates were washed three times with 200 µl of a solution consisting of 30% (v/v) methanol in 35 mM ammonium bicarbonate, pH 8. The final protein concentration was adjusted to 1 µg/µl. Tryptic digestion was carried out with MalS and carboxamidomethylated MalS (1 mg/ml each) in 100 µl 35 mM ammonium bicarbonate solution containing 30% (v/v) methanol, pH 8. A trypsin solution (10 µl; 1 mg/ml; 1 mM HCl) was added to yield a final pH of 7.5 (E:S = 1:10). Samples were incubated at 37 °C for 2 h. Aliquots (0.5 µl) were used for MALDI-MS analysis without further purification. For subsequent HPLC separation the peptide mixture (50 µg) was lyophilized and redissolved in 100 µl of HPLC solvent A.
HPLC Separation of MalS and Tryptic Peptides from MalSA Waters Millipore solvent delivery system, consisting of two HPLC pumps (Waters M510 and Waters M45), was used. Purification of MalS was done using a 250 × 8 mm Grom-Sil 200 Butyl-1 ST reversed phase C-4 column (300 Å, 11 µm) equipped with a Grom precolumn. Separations of the tryptic peptide mixture were carried out using a 250 × 4.6-mm Vydac reversed phase C-18 column (300 Å, 10 µM) equipped with a Vydac precolumn. In all cases, solvent A was 0.1% trifluoroacetic acid in H2O and solvent B was 0.07% trifluoroacetic acid in acetonitrile. For MalS purification the flow rate was adjusted to 2.4 ml/min, and after sample injection, the solvent mixture was kept constant at 10% B for 5 min and was raised to 90% B over a period of 55 min. MalS-containing samples were collected, lyophilized, and redissolved for ESI-MS analysis in 10% acetic acid/2,2,2-trifluoroethanol (7:3), pH 2, to a final concentration of 1-2 µM. For determination of the disulfide bond-containing peptides, 100 µl of the tryptic peptide-containing solution was injected. The flow rate was adjusted to 1 ml/min and the solvent mixture was kept constant at 10% B for 5 min and was raised to 55% B over a period of 45 min. The lyophilized HPLC fractions were dissolved in 5 µl of acetonitrile, 0.1% trifluoroacetic acid (2:1), pH 2, and were used for subsequent MALDI-MS analysis.
Reduction of the Disulfide Bond-containing Tryptic Peptides of MalSFor reduction of the disulfide bond-containing peptides in solution, 1 µl of each HPLC fraction was mixed with 5 µl of 50 mM ammonium bicarbonate solution, pH 8, and a solution of 1 µl of 100 mM 2-mercaptoethanol in H2O was added. The mixture was incubated at 37 °C for 15 min. Aliquots (0.5 µl) were used for MALDI-MS analysis without further purification. For on-target reduction, a solution of 25 mM TCEP in 50 mM ammonium bicarbonate (1 µl; pH 7.5) and acetonitrile (1 µl) was added to the solid matrix/peptide mixture and mixed gently until all solid material was completely redissolved. The reaction mixture adopted pH 4. After 15 min (23 °C) the solvent was evaporated. The matrix/peptide mixture was washed once with 2 µl of 0.1% trifluoroacetic acid, pH 2, and was recrystallized once from 1 µl of acetonitrile, 0.1% trifluoroacetic acid (2:1), pH 2, prior to MALDI-MS analysis.
Mass Spectrometric Molecular Weight Determination and Peptide MappingMatrix-assisted laser
desorption/ionization-time-of-flight-mass spectrometric (MALDI-TOF-MS)
analyses were carried out using a Bruker Biflex time-of-flight mass
spectrometer (Bruker Franzen, Bremen, Germany), equipped with a UV
nitrogen laser (337 nm) and a dual microchannel plate detector. For the
molecular weight determinations, acceleration voltage was set to 25 kV,
and spectra were calibrated with recombinant human
macrophage-colony-stimulating factor (49,030 Da) (37). For peptide
mapping experiments acceleration voltage was set to 20 kV and insulin
was used for calibration. MalS- and tryptic peptide-containing
solutions (0.5 µl) were mixed with 0.5 µl of matrix solution (10 µg/µl 4-hydroxy--cyanocinnamic acid, HCCA) dissolved in
acetonitrile, 0.1% trifluoroacetic acid (2:1), pH 2, directly on the
target. Spectra were recorded after evaporation of the solvent and
processed using the X-MASS data system.
Electrospray ionization-mass spectrometry (ESI-MS) was performed with a Vestec-201A quadrupole mass spectrometer (Vestec, Houston, TX). The ion-spray interface temperature was approximately 45-55 °C for all measurements. The mass analyzer with a nominal m/z range of 2000 was operated at 1/8 unit resolution. An electrospray voltage at the tip of the stainless steel capillary needle of 1.4-1.6 kV and a nozzle-repeller voltage of 20-40 V were employed, respectively. Mass calibration was performed with the 8+ to 12+ charged ions of hen egg white lysozyme (14306.5 Da) and with the 13+ to 17+ charged ions of bovine trypsinogen (23980.9 Da). Raw data were analyzed using a Tecnivent Vector-2 data system. MalS samples were delivered into the mass spectrometer by infusion through a 50-µm (inner diameter) fused silica capillary at a flow rate of 1 µl/min using a Harvard-44 infusion pump.
MalS was purified using a simple procedure involving cold osmotic
shock followed by ion exchange chromatography (Fig.
1A). Purified MalS migrated on
a 10% SDS-PAGE gel under reducing conditions according to its
predicted molecular mass of 74 kDa, and the sample showed homogeneity
(>95%) as no further protein bands were observed.
To determine optimal conditions for assay of the amylase, several
parameters were investigated. The pH optimum of MalS was found to be
between pH 8.0 and 8.5 (Fig. 1B). Therefore, we used Tris-HCl buffer, pH 8.0, in all assays. The kinetic parameters of pure
MalS were determined with PNP6 as substrate by varying substrate
concentrations between 0.5 and 500 µM. The
Km value was 49 µM, and the maximum
velocity (Vmax) was 0.36 µmol min1 mg
1 protein.
Since the activity of many amylases is dependent on Ca2+, we tested whether MalS activity was inhibited by EDTA and whether the inhibitory effect could be reversed by addition of divalent cations. The addition of 1 mM EDTA to the reaction buffer abolished MalS activity. After adding back Ca2+ and Ba2+, enzymatic activity was restored. We detected that Ba2+ even stimulated amylase activity slightly. Although Mg2+ and Mn2+ restored MalS activity to 77 and 74%, respectively, the addition of Zn2+ did not abolish the inhibition by EDTA (Table I).
|
We determined whether conditions of high or low concentrations of salt
would affect MalS activity. MalS activity was highest (350 nmol
min1 mg
1) when no NaCl was added to the
standard assay buffer and decreased with increasing concentrations of
salt. It was about 2-fold lower in the presence of 1 M
NaCl.
MalS migrated at its predicted molecular mass under
reducing conditions, i.e. 74 kDa. Under nonreducing
conditions, MalS migrated near 90 kDa. Furthermore, the same shift from
74 to 90 kDa was detected after reoxidation of reduced MalS using
oxidized glutathione, indicating the presence of disulfide bonds (Fig.
2A). However, since proteins
containing disulfide bonds normally migrate at lower apparent
molecular weight than under reducing conditions, we investigated
the possibility that the unusual behavior of MalS may be due to other
explanations, e.g. other post-translational modifications.
ESI-MS molecular weight analysis of nonreduced MalS showed a series of multiply charged molecular ions centering around the [M + 43H]43+ molecular ion (Fig. 2B), indicating the homogeneity of the sample. The experimentally determined molecular mass of 73,978 Da (±20) was in excellent agreement with the calculated molecular mass (73961 Da) from the translated amino acid sequence; thus, the existence of a covalent multimeric form of MalS or other post-translational modifications besides possible disulfide bond formation could be ruled out. In addition, iodoacetamide alkylation of nonreduced MalS did not yield carbamidomethylated product, indicating the absence of free cysteine residues (data not shown).
Peptide Mapping Using Reduced MalSTo further investigate the molecular structure of MalS, the reduced and iodoacetamide alkylated protein was cleaved proteolytically with trypsin, and the fragment mixture was subsequently analyzed by MALDI-MS (mass spectrometric peptide mapping). The results (Table II) confirmed the amino acid sequence (Fig. 3) as very good sequence coverage was obtained. Only in the cases of partial sequences with closely spaced lysine or arginine residues were we not able to identify the resulting short peptides directly from peptide mapping data due to superimposition with matrix ions. However, the correct N terminus (amino acids 1-27; fragment T1) was ascertained by an ion signal at m/z 2837, and the C-terminal tryptic peptide (amino acids 651-659) of the protein was identified as an ion signal at m/z 1000. Cysteine-containing peptide ions were observed, e.g. at m/z 2461 for carboxamidomethylated T2 and at m/z 2172 for carboxamidomethylated T3 (Table II).
|
Disulfide Bond Mapping
To map disulfide bonds, MalS was
digested with trypsin prior to reduction. Digestion was carried out at
pH 7.5 at 37 °C but was limited to 2 h to minimize possible
disulfide bond scrambling (14, 15, 38). MALDI-MS peptide mapping showed
comparable spectra as with reduced MalS, but additional ion signals
were observed that could not be addressed as tryptic peptides but as disulfide-bonded dipeptides at m/z 4515, assignable as T2-S-S-T3; at m/z 2544, assignable as
dipeptide T7-S-S-T42; and at m/z 2586, assignable as
dipeptide T3-S-S-T7, respectively. As more than two possibly
disulfide-linked peptide ion signals were detected in the peptide
mapping experiments, and to reliably distinguish between the two
possibilities T7-S-S-T42 and T3-S-S-T7, which gave only weak ion
signals, the tryptic peptide mixture was fractionated by HPLC. MALDI-MS
analyses were carried out on all fractions to identify the peptides
(Table II, Fig. 4) and showed that
separation of the complex peptide mixture was incomplete, and in nearly
all fractions two or more peptides coeluted. Interestingly, the
peptides with m/z 2544 and
m/z 2586 coeluted in one HPLC fraction and could only be separated by 2-fold rechromatography (cf. Fig.
4C). The HPLC fractionated peptide with
m/z 4515 (Fig. 4A) was reduced separately in ammonium bicarbonate solution, and MALDI-MS analysis was
repeated (Fig. 4B). In addition to the complete
disappearance of the dipeptide ion signal at m/z
4515, two new strong ion signals were observed upon reduction at
m/z 2404 which corresponds to T2 and
m/z 2114 corresponding to T3 proving the presence
of the disulfide bond Cys40-Cys58. By
contrast, the disulfide-linked dipeptide T7-S-S-T42
(m/z 2544) was reduced with TCEP on the MALDI
target in the presence of the HCCA matrix at pH 4. TCEP was used for
these experiments as this reducing agent is applicable even at acidic
pH (39). The ion signal at m/z 2544 disappeared,
although not completely, and two new strong ion signals at
m/z 2070 (T42) and 2259 (T42-HCCA adduct) were
observed. An ion signal for T7 (m/z 477) was not observed due to suppression of the ion. However, the mass difference of
474 (3 mass units lower than calculated due to three additional protons
added by reduction and protonation) between the ion signal at
m/z 2544 for the disulfide-linked dipeptide and
the ion signal with m/z 2070 for T42 indicated
the existence of T7 in this fraction. Thus, it can be concluded that
mature MalS contained two disulfide bonds linking
Cys104-Cys520 and
Cys40-Cys58 (cf. Fig. 3 and Fig.
4). The ion signal at m/z 2586 resisted reduction
and, thus, did not represent a disulfide-linked peptide. As this ion
signal does not correlate to a tryptic peptide, it remains
unassigned.
Expression of MalS in the Cytoplasm
To obtain initial evidence for the importance of disulfide bonds for proper folding of MalS, we asked whether MalS can fold into its active conformation when expressed in the reducing environment of the cytoplasm. When we expressed signal sequenceless MalS from pCS7 in wild-type cells, it was enzymatically inactive (Table III). Since alkaline phosphatase, which is known to require disulfide bonds for enzymatic activity (40), does not fold properly in the cytoplasm of wild-type cells, but can be actively expressed in the cytoplasm of trxB and gsh mutants (41), we tested whether the same result could be obtained for MalS. gsh encodes glutathione synthase which, like thioredoxin reductase (TrxB), is thought to be involved in maintaining a reducing environment in the cytoplasm. However, in all strains tested, MalS was inactive when expressed in the cytoplasm. In contrast to alkaline phosphatase, MalS could not fold into an active conformation even in trxB or gsh mutants (Table III). The absence of MalS activity could be correlated to its inability to form disulfide bonds since only reduced MalS migrating at 74 kDa was detected under nonreducing conditions on SDS-PAGE (Fig. 5).
|
MalS Activity under Reducing Conditions
In contrast to the
above results, the activity of native MalS under reducing conditions
indicates that disulfide bonds are not required for MalS function. At
room temperature, properly folded MalS was active in standard assay
buffer containing either 0.01, 0.1, 1, 10, or 30 mM DTT. In
the absence of DTT amylase activity was 292 nmol min1
mg
1. In the presence of the given DTT concentrations it
was 330 ± 30 nmol min
1 mg
1. To
confirm that disulfide bonds were reduced by the addition of DTT, we
determined the migration of MalS on SDS-PAGE. MalS migrated at 74 kDa
after the addition of DTT indicating that disulfide bonds were reduced
(Fig. 2A). It should be noted that samples were not
heat-treated prior to electrophoresis. Also, mass spectrometric peptide
mapping analyses of reduced MalS confirmed that both disulfide bonds
were reduced completely by using 200-fold molar access of DTT and could
be alkylated quantitatively with iodoacetamide (see above).
Disulfide bonds enhance the thermal stability of many proteins (42-45). Since we detected no loss of MalS activity under reducing conditions, stability of MalS toward thermal inactivation was investigated under reducing and nonreducing conditions, i.e. in the presence and absence of DTT. As determined by MalS assays, stability of reduced MalS was only slightly less than that of oxidized MalS (Table IV). A complete loss of MalS activity was observed at 61 °C under nonreducing conditions as well as under reducing conditions. Similar results were obtained when thermal denaturation of MalS was assayed in whole cells. In this case, MalS activity was abolished at 55 °C under nonreducing conditions as well as under reducing conditions (data not shown).
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SDS resistance has been reported for a fungal -amylase (46).
However, MalS was shown to be sensitive to SDS. Half-maximal activity
was detected near 0.2% SDS (Table V).
Subsequently, we tested whether the presence of disulfide bonds would
enhance the tolerance of MalS toward SDS. Only at SDS concentrations
above 0.25% was the difference in enzymatic activity between reduced and oxidized MalS 50% or greater.
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The
analysis of the MalS protein by sedimentation equilibrium
ultracentrifugation yielded a molecular mass of 70 ± 4 kDa under oxidizing conditions. This was in agreement with the calculated molecular weight within the error of the measurement. The fit of the
data to Equation 1 is very good and shows no signs of the presence of
additional species, as can be deduced from the residuals of the fit
which show a random scattering around the fit curve. Thus, the results
demonstrate unambiguously that the protein is present as a monomer at
concentrations up to the µM range (Fig. 6).
Electron Microscopy
Electron microscopy of purified MalS was
used to obtain indications about the structure of MalS under
nonreducing and reducing conditions. MalS is a globular protein with
the shape of a horseshoe (Fig. 7). Using
negatively stained samples, the height could be determined to 80.5 Å ± 0.88. The complete diameter was 57.5 Å ± 0.63, and the inner
diameter of the hollow space was 23.0 Å ± 0.43.
After incubation with DTT, MalS aggregated to form large clumps (Fig. 7C). Thus, reduction of disulfide bonds lead to at least partial denaturation of MalS. It could be speculated that hydrophobic segments may be exposed at the surface causing intermolecular interaction. Since no loss of enzymatic activity was detected under these conditions, the observed partial denaturation did not affect the active site.
MalS differs from the numerous amylases identified and
characterized to date. MalS has an N-terminal extension of about 160 amino acids that is not homologous to other amylases but rather shows
homology to the proposed peptide binding domain of GroEL (Fig.
8). The function of the MalS N terminus,
which is linked via a disulfide bond formed between Cys104
and Cys520 to the C-terminal amylase domain, could be to
assist in folding of the amylase domain. Reduction of disulfide bonds,
which most likely leads to detachment of the N and C termini of MalS,
caused aggregation. Since one of the main functions of molecular
chaperones is to prevent aggregation, it is tempting to speculate on an
autochaperone activity of the MalS N terminus.
MalS contains a second disulfide bond formed between Cys residues 40 and 58, located in its N-terminal domain. Of the Cys residues involved in disulfide bond formation, only Cys520 is conserved in a number of amylases, e.g. of Alteromonas haloplanctis (47), Aspergillus shirousamii (48), Schwanniomyces occidentalis (49), and Saccharomycopsis fibuligera (50), where this residue, together with other conserved Cys residues, is involved in disulfide bond formation within the amylase domains. However, Cys40, Cys58, and Cys104 seem to be uniquely present in MalS. This may explain that MalS was active even after reduction of disulfide bonds.
Initial evidence for the importance of disulfide bonds for folding of
MalS was obtained by expression of MalS under reducing conditions in
the cytoplasm, which yielded inactive protein. The inability of MalS to
become active in the cytoplasm, even when the trxB and
gsh mutations were present, was surprising since several
periplasmic proteins have been shown to be actively expressed in the
cytoplasm; examples include maltose binding protein (51), alkaline
phosphatase (41), -lactamase (52), and trehalase TreA.2 It could be argued
that the inability of signal sequenceless MalS to fold into its active
conformation in the cytoplasm may be an artifact caused by the genetic
manipulation of signal sequence removal. This explanation can be
excluded since it was shown earlier that this MalS construct could be
functionally expressed in the periplasm when exported by an altered
secretion machinery, i.e. in prlA mutants
(23).
Locating cystine bridges in proteins generally involves cleavage of the protein by enzymatic or chemical means under which disulfide scrambling is avoided. However, with nondenatured and disulfide bond containing proteins, nonspecific or incomplete proteolytic cleavages are frequently observed (e.g. Refs. 15 and 37). Therefore, cleavage by trypsin was first carried out with irreversibly denatured, i.e. reduced and carboxamidomethylated, MalS to study specific cleavage products. Disulfide bonds were destroyed in this sample, but sequence verification was possible as almost all predicted peptide ions were observed (cf. Table II). Using this information, reversibly denatured MalS (by addition of 30% methanol (37)) was digested with trypsin for 2 h at pH 7.5, a rather short cleavage period, but these conditions were chosen to minimize disulfide bond scrambling (15). Subsequently, disulfide bonds in MalS were identified by mass spectrometric analyses.
To determine whether or not an observed peak in the recorded mass spectrum is due to disulfide linkages, the corresponding ion signal should completely, or at least mostly, be eliminated by reduction. This is important as the recently discovered MALDI-induced cleavage of disulfide bonds (53-55), which was also observed in our analyses (cf. Fig. 4, A and C), may obscure the results, particularly when peptide mixtures are analyzed. Thus, chemical reduction either in solution or on-target, as demonstrated here, is necessary to address disulfide-linked dipeptides unambiguously. Whereas reduction in solution (at pH 8) led to complete reduction of the disulfide-linked peptides, reduction on the MALDI target in the presence of the matrix (at pH 4) was incomplete and additionally formed strong peptide-matrix adduct ions (Fig. 4D). The presence of TCEP on the target did not interfere with peptide ion detection. Thus, this strategy proved successful for identification of disulfide bonds even with very little material, e.g. after several chromatographic separation steps.
Further information on the structure of MalS was obtained by electron
microscopy and analytical ultracentrifugation. The EM data indicated
that MalS is present as a U-shaped structure which looked similar to
amylopullulanase (56) and a glycoamylase of Clostridium
thermosaccharolyticum (57). The latter is also a monomer as has
been determined by analytical ultracentrifugation (58). A topological
model of the MalS protein was derived from these structural data (Fig.
9). We propose that MalS is composed of
at least two domains, an N-terminal extension carrying a disulfide bond
and a C-terminal amylase domain that is connected to the N-terminal
segment via the second disulfide bond. The presence of extensions was
reported for other amylases; however, these extensions are not
N-terminal, are not connected via a disulfide bond to the amylase
domain, and do not exhibit homology to the relevant domains of MalS or
GroEL (59-61).
We are grateful to Winfried Boos and Michael Przybylski in whose laboratories parts of this work was carried out. We thank Ann Flower for reading the manuscript.