(Received for publication, December 10, 1996, and in revised form, February 6, 1997)
From the ¶ Institut für Biochemie, Universität
Witten/Herdecke, Stockumer Str. 10, 58453 Witten, Germany, the
Institut für Physiologische Chemie I,
Ruhruniversität Bochum, Universitätsstr. 150, 44780 Bochum,
Germany, and
bitop GmbH Witten, Stockumer Str. 10, 58453 Witten, Germany
After developing a suitable procedure to produce
large amounts of Euglena gracilis as well as a reliable
protocol to purify the multifunctional tryptophan-synthesizing enzyme
derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995)
Biotechnol. Appl. Biochem. 22, 179-190), we here describe
structural and catalytic properties of the multifunctional
tryptophan-synthesizing enzyme. The kinetic parameters
kcat of all five activities and
Km for the main substrates were determined. The
relative molecular weight under denaturing conditions as judged by
SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as
well as gel filtration experiments revealed that the enzyme exists as a
homodimer. Neither intersubunit disulfide linkages nor glycosylations
were detected. On the other hand, the polypeptide chains are blocked
N-terminally. Complete tryptic digestion of the protomer, high pressure
liquid chromatography separation of the resulting peptides, and
N-terminal sequence analysis of homogenous peaks as judged by
matrix-assisted laser/desorption ionization time-of-flight mass
spectrometry was performed. Depending on the sequenced peptides,
alignments to all entries of the SwissProt data base resulted in both
strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using
endoproteinase Glu-C uncovered one major cleavage site yielding a
semistable, N-terminally blocked fragment with a molecular weight of
119,000. In addition, an increase in -elimination accompanied by a
decrease in
-replacement activity of the
-reaction during
proteolysis was observed.
The terminal five reactions of the biosynthetic pathway yielding
L-tryptophan are catalyzed by
anthranilate-5-phosphoribosyl-1-pyrophosphate phosphoribosyltransferase
(PRT1; EC 2.4.2.18),
phosphoribosylanthranilate isomerase (PRAI; EC 5.3.1.24),
indole-3-glycerol-phosphate synthase (IGPS; EC 4.1.1.48), and
tryptophan synthase (- and
-chains, EC 4.2.1.20, see Scheme
I).
In most procaryotes, tryptophan synthase is organized as a tetrameric
bienzyme complex consisting of a central 2-dimer
(TRPS
) combined with two peripheral
-subunits (TRPS
). The
Escherichia coli and Salmonella typhimurium
enzymes in particular have been intensively investigated in the past
(1, 2). In fungi such as Saccharomyces cerevisiae and
Neurospora crassa, tryptophan synthase is a dimer of
identical bifunctional polypeptides (3, 4, 5). In contrast, the enzyme
isolated from Euglena gracilis, here designated as
multifunctional tryptophan-synthesizing enzyme (MTSE), is capable of
catalyzing all reactions shown in Scheme I. Although this discovery was
made 2 decades ago (6, 7), no thorough further characterization of this
multifunctional enzyme has been published so far, probably due to the
excessive expenditure to cultivate sufficient amounts of E. gracilis and the difficulties of purifying MTSE with outdated
technology. Using state of the art equipment and materials, we
established a reliable and simple procedure to obtain MTSE from
E. gracilis (8). Here we present both a reinvestigation of
earlier initial findings and new data with respect to structural and
functional properties of the enzyme.
If not otherwise
stated, all chemicals (reagent or ultrapure grade) were obtained from
Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany), and all
enzymes were from Boehringer (Mannheim, Germany). Pyridoxal
5-phosphate was from Serva (Heidelberg, Germany), and phosphoribosyl
pyrophosphate was from Sigma (Deisenhofen, Germany).
Indoleglycerol 3-phosphate (IGP), phosphoribosylanthranilate (PRA), and
o-carboxyphenylaminodeoxyribulose 5-phosphate (CDRP) were synthesized as described earlier (9-11). E. gracilis
MTSE was prepared from algal biomass as described previously (8). Quartz-bidistilled water was used throughout. E. coli
strains W3110 trp C 9830 (F
) and W3110 trp D 9923 (D
) were a generous gift from Dr. Charles Yanofsky.
PRT activity at substrate
concentrations below 75 µM was determined
fluorimetrically (12), whereas at higher substrate concentrations, a
spectrophotometric assay at 278 nm was performed (11). Assays were
performed both in the presence and in the absence of 0.1 mg/ml E. coli crude cell extract complementing consecutive "helper" activities but lacking PRT (W3110 trp D 9923 (D)).
PRAI activity was determined spectrophotometrically at 278 nm using a
0.1 mg/ml concentration of a PRAI-deficient E. coli helper
cell extract (W3110 trp C 9830 (F)) (11).
To determine IGPS activity both a spectroscopic (13) and a fluorimetric assay system (14) were used.
The activity of tryptophan synthase was measured using an
NADH-coupled assay system (11), and the formation of
L-tryptophan from L-serine and indole
(
-replacement,
-reaction) was determined as described earlier
(15).
The overall activity of MTSE was measured in 20 mM
Tris/HCl, pH 7.8, containing 0.05 mM anthranilate, 1 mM phosphoribosyl pyrophosphate, 50 mM
L-serine, 2 mM MgSO4, 0.04 mM pyridoxal 5-phosphate, 0.01 mM
dithioerythritol, 0.25 mM EDTA, 0.025 mg/ml BSA. The change in OD was monitored at 290 nm.
Deamination of L-serine (-elimination,
-side
reaction) was measured using a lactate dehydrogenase/NADH-coupled assay
system as described previously (16).
Calculations to determine Km and kcat were carried out applying a computer program that iterates 20 times an algorithm described previously (17). All measurements were performed in 20 mM Tris/HCl, pH 7.8, at 37 °C, and the reactions were started by adding 0.04 µg of purified E. gracilis MTSE. Fluorimetric assays were carried out using an Aminco Bowman series 2 Luminescence Spectrometer (Sopra, Büttelborn, Germany), whereas spectrophotometric measurements were performed on a UV-2100 spectrophotometer (Shimadzu, München, Germany).
SDS-Polyacrylamide Gel ElectrophoresisSDS-PAGE was
performed on 7.5% acrylamide slab gels using 0.05 µg of MTSE per
slot and myosin, -galactosidase, phosphorylase b, bovine
serum albumin, and ovalbumin as molecular weight markers (18). Runs
under nonreducing conditions were performed in the absence of
-mercaptoethanol in the sample buffer. Gels were stained with
Coomassie Blue G-250 and scanned with an MPS 940.800 densitometer (Vitatron, Rösrath, Germany).
To determine the native molecular weight of MTSE from E. gracilis, 0.2 mg of the enzyme was cross-linked for 10 h at 4 °C in 1 ml of 20 mM potassium phosphate, 2% glutaraldehyde (19). The reaction was stopped by adding 4-fold concentrated SDS-PAGE sample buffer, and the molecular weight of the cross-linked protein was estimated by SDS-PAGE under reducing conditions.
Analytical Gel FiltrationAnalytical gel filtration was
performed on a Superose 6/HR 10/30 column (Pharmacia Biotech, Uppsala,
Sweden), calibrated with thyroglobulin, apoferritin, -amylase,
alcohol dehydrogenase, albumin, carbonic anhydrase, and cytochrome
c (20). 0.5 mg of MTSE was applied to the column, and
elution was performed at 400 µl/min. To avoid unspecific hydrophobic
or ionic interactions between the stationary phase and the proteins,
150 mM NaCl and 10% ethylene glycol were added to the
elution buffer. To follow subunit dissociation, additional runs were
performed using the same calibrated column at increasing concentrations
of guanidinium hydrochloride.
0.1 mg of MTSE from E. gracilis was applied to a glycoprotein assay (21) using the glycoprotein detection kit from Boehringer.
Protein EstimationProtein concentrations were determined according to Bradford (22) using bovine serum albumin as the protein standard.
Tryptic Digestion and N-terminal Sequence AnalysisTo
determine parts of the amino acid sequence, complete tryptic digestion
of E. gracilis MTSE was performed. For that purpose, 100 µl of 5 M guanidinium hydrochloride, 30 mM
dithiothreitol, 0.5 M Tris/HCl, pH 8.6, were added to 150 µg of the purified enzyme and stored at 4 °C under argon
overnight. After unfolding, the sample was dialyzed against 2 liters of
50 mM NH4HCO3, 1 mM
CaCl2, pH 7.8, for 4 h. After dialysis, 6 µg of
trypsin were added, the solution was made 5% (v/v) CH3CN,
and digestion was carried out at 37 °C overnight. After cleavage,
tryptic peptides were separated by reversed phase HPLC using a 140 B
inert pump (Applied Biosystems, Weiterstadt, Germany), an SPD 10 A
detector (Shimadzu, München, Germany), and an MBO-25-05 column
(250 × 0.8 mm, C18, 5 µ, 300 A, LC-Packkings, Amsterdam, The
Netherlands). A linear gradient from 0.1 to 0.09% trifluoroacetic acid
and 0-84% CH3CN was run for 200 min at a flow rate of 10 µl/min, and peaks were detected at 215 nm as well as 295 nm.
Depending on the elution profile, peaks were collected manually.
Symmetrical peaks without visible contaminations as judged by the HPLC
elution profile were further analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-MS) as
described (23) using a Lasermat 2000 (Finnigan MAT). For that purpose,
an aliquot of 0.5 µl from 20 selected peak fractions of the HPLC
separation was mixed with 1 µl of -cyanohydroxycinnamonic acid,
and 1 µl thereof was subjected to MALDI-MS. Seven homogenous peaks,
as judged by MALDI-MS, were subjected to automated Edman degradation
(24, 25), and their N-terminal amino acid sequences were determined
using a 473A or 476A protein sequenator (Applied Biosystems Inc.,
Foster City, CA).
Limited proteolysis was carried out using 0.04 mg/ml of E. gracilis MTSE dialyzed against 50 mM Tris/HCl, pH 7.8, at 37 °C and 7 µg/ml endoproteinase Glu-C from S. aureus. After different time intervals, samples were taken, assayed for enzymatic activities, and (after boiling for 5 min in SDS-PAGE sample buffer) subjected to SDS-PAGE analysis.
To elucidate the flux of the respective metabolites in MTSE, the Km and Vmax values of the involved active centers for their respective substrates were measured. A summary of the kinetic data is given in Table I.
|
Using anthranilate as the substrate, PRT activity was measured both in the presence and in the absence of complementing helper activities, and no difference was observed. Steady state kinetics reveal significant substrate inhibition at anthranilate concentrations higher than 50 µM. The data indicate a slight cooperativity. Nevertheless, for better comparability a K0.5 value was calculated according to the Michaelis-Menten model.
PRAI activity, if determined without an E. coli helper extract, was about 30% lower than the value obtained in the presence of the cell extract. An influence on Km could not be detected. PRAI activity showed Michaelis-Menten behavior, and the enzyme was inhibited competitively by anthranilate with a Ki value in the millimolar range (data not shown).
Kinetic measurements with IGPS resulted in good data fits to the Michaelis-Menten model. The Km of MTSE for CDRP is much higher than the Km for anthranilate and PRA.
Michaelis-Menten behavior was observed for TRPS activities with IGP
as substrate. No inhibition at higher anthranilate concentrations could
be detected.
Depending on the substrate, measurements of TRPS activities revealed
a different picture. Whereas titration with indole resulted in
significant cooperativity, conversion of L-serine obeyed
the Michaelis-Menten model.
In addition to the described single enzymatic activities, the overall
reaction (anthranilate L-tryptophan) was investigated using a spectrophotometric assay system at 290 nm in the presence of
all necessary cosubstrates, phosphoribosyl pyrophosphate, and L-serine. The overall reaction was inhibited by
anthranilate. In contrast to the Km values,
kcat values of all activity measurements were in
the same range.
Measurements of the overall reaction at different concentrations of
potassium phosphate, sodium chloride, and Tris/HCl clearly showed an
activity optimum at a concentration of 40 mM for these salts. In contrast, measurements at increasing concentrations of
ammonium sulfate resulted in slightly increasing activities, but no
distinct maximum was detectable (Fig. 1). The pH optimum was 7.8, and the temperature optimum was at 52 °C (data not
shown).
Molecular Weight Estimation and Subunit Composition
The relative molecular weight of the purified enzyme and the product of cross-linking by glutaraldehyde, respectively, was determined by both reducing and nonreducing SDS-PAGE. The relative subunit molecular weight is 136,000, whereas the cross-linked chains migrated to a position corresponding to a value of 272,000 (data not shown). Calibrated gel filtration of the native enzyme using a Superose 6/HR column yielded a relative molecular weight of 245,000 (data not shown).
To follow subunit dissociation, analytical gel filtration was performed
using buffers containing different concentrations of guanidinium
hydrochloride. As shown in Fig. 2 (inset),
two peaks in the elution profile can be detected at 1.2 M
guanidinium hydrochloride, indicating the presence of both dimeric and
dissociated protein species. Since it is well known that guanidinium
HCl-induced subunit dissociation is followed by further unfolding of
the resulting monomers at higher concentrations of the chaotropic
agent, the elution volume in gel filtration decreases as a
consequence.
Partial Primary Structure and Posttranslational Modifications
Using the Boehringer glycosylation detection kit,
no glycosylation of MTSE from E. gracilis could be detected
(data not shown). Several approaches of N-terminal sequence analysis of
the native enzyme failed, indicating that the protomer is blocked
N-terminally. To obtain information about the primary sequence,
complete tryptic digestion followed by reverse phase HPLC was
performed, and seven homogenous HPLC peaks as determined by MALDI-MS
were subjected to automated N-terminal sequence analysis making use of
Edman degradation. In addition to the resulting seven main sequences, impurities within four HPLC peaks that were not detected by MALDI-MS yielded additional sequences. The resulting sequences are summarized in
Fig. 3.
A similarity search was performed with all entries of the SwissProt
protein sequence data base release 32.0 using the BLASTP program (26)
and the BLOSUM62 scoring matrix (27). Whereas peptides 2a, 3a, 4b, 6a,
and 7b exhibited no sequence homology to known Trp sequences, peptides
1a, 4a, 5a, and 7a showed homologies to PRT (TrpD), and peptides 5b and
6b showed homologies to IGPS (TrpC) and TRPS (TrpB), respectively.
Alignments of the sequenced peptides to Trp sequences as obtained by
BLASTP are given in Fig. 4.
Limited Proteolysis
Limited proteolysis using Endoproteinase Glu-C revealed an intermediate fragment with a relative molecular weight of 119,000 that is slowly degraded further during the time course of proteolysis (data not shown). The corresponding Mr 17,000 fragment could not be detected, indicating its rapid further degradation, possibly due to a loss in conformational protection against proteolytic attack. N-terminal sequence analysis of the Mr 119,000 fragment by Edman degradation failed.
To shed some light on the effect of proteolytic cleavage by Endoproteinase Glu-C, we determined both the activity and the reaction specificity of E. gracilis MTSE. During the time course of proteolysis, a loss in tryptophan synthase activity accompanied by an increase in serine deaminase activity was observed (data not shown).
The turnover numbers
of both the overall activity and all five single activities of E. gracilis MTSE are in the same range, indicating that none of the
single activities comprises a rate-limiting step in the overall
reaction. Although slightly different, the kcat
values reported here roughly correspond to the specific activities as
determined by Hankins and Mills (7). Table II gives an
overview of turnover rate constants of tryptophan pathway enzymes from other sources. However, care should be taken in directly comparing those values, since various parameters such as buffer composition or
reaction temperatures were different in the respective investigations. Nevertheless, compared with the respective enzymes from other sources,
it can be seen that kcat of PRT, TRPS, and
TRPS
of E. gracilis MTSE are the lowest values, whereas
kcat of PRAI and IGPS lie in a medium range.
Thus, with respect to the single turnover rates, E. gracilis
MTSE has no catalytic advantage compared with the individual enzymes
derived from other microorganisms. Regarding the overall pathway from
anthranilate to L-tryptophan within the living cell, we
conclude that due to a decreased diffusion barrier even the low
turnover rates of PRT and tryptophan synthase within E. gracilis MTSE are sufficient to result in a comparable overall efficiency to synthesize L-tryptophan.
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Comparing the Km values for all reaction intermediates from anthranilate to L-tryptophan gives an interesting pattern: the Km values for the first and the last intermediates of the reaction pathway catalyzed by E. gracilis MTSE (anthranilate, PRA, and indole) are smaller by more than 1 order of magnitude as compared with those in the middle of the pathway (CDRP and IGP). This is exactly what one would expect if substrate channeling via a tunnel is realized in E. gracilis MTSE. Reaction intermediates, binding to active centers located in the core of the enzyme need to overcome a greater diffusion barrier. Substrate channeling is known for tryptophan synthase in S. typhimurium and E. coli (78-81) but not for PRAI/IGPS in E. coli (82). However, further detailed kinetic data are necessary to allow conclusions regarding substrate channeling in E. gracilis MTSE.
Molecular Weight of E. gracilis MTSEDetermination of the subunit molecular weight of E. gracilis MTSE by MALDI-MS failed, since no suitable desorption conditions could be established. Nevertheless, SDS-PAGE analysis of the denatured and of the cross-linked protomer revealed a relative molecular weight of 136,000 and 272,000, respectively. The fact that densitometry of the respective gel yields only one highly symmetric band provides strong evidence that the enzyme exists as a homodimer of 272,000. However, the possibility of the existence of a heterodimer consisting of chemically different but physically similar chains cannot be totally excluded.
The values found in this investigation do not correspond to earlier
reports making use of native molecular weight estimations by gel
filtration over Sephadex G-200 as well as density gradient centrifugations (234,000; Ref. 6) and are even in contradiction with
refined measurements (325,000 and 155,000 for the native enzyme and the
subunit respectively; Ref. 7). However, we believe that our estimation
resulted in the most accurate value, since the migration behavior of
E. gracilis MTSE as shown in the original publication of
Hankins and Mills (7) is identical to that observed in our experiments.
The discrepancy obviously is due to a wrong calibration based on a
false assumption of the molecular weight of -galactosidase
(150,000), one of only three molecular weight markers in their
experiment. The molecular weight of
-galactosidase was later
determined to be 118,000 (83). Compared with SDS-PAGE of cross-linked
enzyme multimers, native molecular weight estimations using gel
filtration in general are less accurate. In the latter case, the
dependence on molecular shape as well as possible retardations caused
by ionic and/or hydrophobic interactions with the stationary phase
further increases possible errors. Indeed, we observed hydrophobic matrix interactions in the absence of ethylene glycol (data not shown),
and even under improved buffer conditions the molecular weight under
native conditions as determined by gel filtration (245,000) is smaller
than expected, indicating the presence of significant residual
retardation effects. We exclude the possibility that the enzyme under
those conditions exists as a monomer, because the addition of
guanidinium hydrochloride clearly demonstrated a transition to monomers
requiring a concentration of 1.2 M (Fig. 2).
Table III gives a summary of the molecular weights of
all monofunctional Trp enzymes catalyzing reactions that are also
catalyzed by E. gracilis MTSE. A polypeptide chain
consisting of five domains homologous to the above mentioned subunits
requires an average molecular weight of 164,771 (Table III, means).
Even if the smallest molecular weight values ever published of each
single subunit are considered, the minimum sum is still 152,495 (Table
III,
smallest values). Evidently, this sum is considerably higher
than the molecular weight of E. gracilis MTSE (136,000).
Thus, simple gene fusions of all five genes as known for the gene
corresponding to bacterial trpA and trpB in
S. cerevisiae (59), N. crassa (103), or
Corprinus cinereus (56) can be excluded. Moreover, gene
fusion would require additional sequences acting as connectors, resulting in an even higher molecular weight. Thus, either a
considerably different architecture is realized, or one of the active
centers possibly catalyzes a combined reaction e.g.
isomerization of PRA to CDRP followed by IGP synthesis. The structural
scaffold of three subunits (PRAI, IGPS, TRPS
) has a
(
/
)8 barrel topology. Although the sequence homology
between PRAI and IGPS is rather low, they are structurally very similar
so that they can be superimposed with a root mean square deviation of
2.03 Å for 138 C
pairs (104). Thus, it would be
conceivable that one domain catalyzes both PRA isomerization and IGP
synthesis. This argument is corroborated by the finding that the
Km value of the respective active center for CDRP is
rather high, meaning that CDRP might not be a true intermediate in
Euglena MTSE. Consequently, assuming two instead of three
TIM barrels in E. gracilis MTSE reduces the expected molecular weight by 27,982, the size of an average TIM barrel enzyme
(Table III, mean of TIM barrel means). The resulting expected molecular
weight would then be 136,789 (Table III,
means
mean of TIM
barrel means), a value almost identical to the molecular weight of
E. gracilis MTSE as determined in this investigation. However, since MTSE is so far the only enzyme exhibiting all activities based on the (
/
)8 barrel motif within one polypeptide
chain, no comparison with known species can be made.
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Alignment of
the sequences of the 11 peptides to all SwissProt entries resulted not
only in similarities to known Trp sequences but interestingly also in
five sequences with no homology at all. This finding indicates unknown
parts of the primary structure of E. gracilis MTSE that may
belong to regions connecting different domains. Homology to IGPS was
observed for peptide 5b. In the three-dimensional structure, this part
of the sequence corresponds to the 4-
5 loop, helix 5, and strand
5 of the essential TIM barrel fold as described for IGPS from E. coli (104) and Sulfolobus solfataricus (75). Thus, we
are inclined to expect a similar fold in the E. gracilis
enzyme. Peptide 6b is homologous to the N-terminal region of the
bacterial
-subunit, which in S. typhimurium has no
defined secondary structure and is located at the contact surface to
the
-subunit (105). Assuming that this contact depends on this
highly conserved region (Fig. 4), it is conceivable that, as in
bacterial tryptophan synthase, the
- and
-domain of the E. gracilis enzyme are in contact. Four peptides are homologous to
PRT. However, three-dimensional structures of PRT from different sources are not yet known.
Limited
proteolysis is an ideal tool to detect solvent-exposed loops lacking
defined secondary structure as described for the hinge region in the
-subunit of tryptophan synthase from E. coli (106) or for
the connector region of tryptophan synthase from S. cerevisiae (86). Using endoproteinase Glu-C (and some other
endoproteinases; data not shown), only one major cleavage site could be
detected. This finding gives evidence that either only one connector in
E. gracilis MTSE is present or that possible other
connecting peptides are not susceptible to proteolytic attack under the
given experimental conditions.
Limited proteolysis of E. coli TRPS, using endoproteinase
Glu-C results in a shift from
-replacement to
-elimination
specificity and can be expressed by the ratio of tryptophan synthase to
serine deaminase activity (44). Consequently, we measured both
tryptophan synthase and serine deaminase activities of MTSE during the
time course of proteolysis. Albeit less significant, we observed the same shift in reaction specificity as described for the E. coli enzyme. Therefore, it is likely that proteolytic cleavage
occurs near or within the
-domain of E. gracilis MTSE.
The difference between the molecular weight of native MTSE and the
large fragment after proteolysis roughly corresponds to the smaller
(F2) fragment after limited proteolysis of the
-subunit of
tryptophan synthase from E. coli. Thus, proteolytic cleavage
is likely to occur in a structural element corresponding to the hinge
region in E. coli. Since N-terminal sequence analysis of the
larger proteolytic fragment failed, we assume that this fragment
contains the blocked N terminus. Thus, the site of proteolytic
cleavage, and possibly the
-domain itself, may be located near the C
terminus.
For three reasons, E. gracilis MTSE is probably not simply the product of gene fusions of all five trp genes corresponding to the five detectable activities: 1) The subunit molecular weight of the purified enzyme is too small, 2) there are new amino acid sequences (no homologies detected by BLASTP), and 3) only one of four postulated connecting peptides can be defined by limited proteolysis. Future work, cloning the MTSE-encoding gene from E. gracilis by using degenerate PCR primers based on the presented amino acid sequences should yield the complete sequence and help to solve this structural puzzle.
Dedicated to Dr. Charles Yanofsky on the occasion of his 72nd birthday.
The expert technical assistance of A. Böhm, H. Korte, and G. Becker is gratefully acknowledged.