From the Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan
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ABSTRACT |
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T-protein is a component of the glycine cleavage
system and catalyzes the tetrahydrofolate-dependent
reaction. To determine the folate-binding site on the enzyme,
14C-labeled methylenetetrahydropteroyltetraglutamate
(5,10-CH2-H4PteGlu4) was
enzymatically synthesized from methylenetetrahydrofolate
(5,10-CH2-H4folate) and
[U-14C]glutamic acid and subjected to cross-linking with
the recombinant Escherichia coli T-protein using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length
cross-linker between amino and carboxyl groups. The cross-linked
product was digested with lysylendopeptidase, and the resulting
peptides were separated by reversed-phase high performance liquid
chromatography. Amino acid sequencing of the labeled peptides revealed
that three lysine residues at positions 78, 81, and 352 were involved
in the cross-linking with polyglutamate moiety of
5,10-CH2-H4PteGlu4. The comparable
experiment with 5,10-CH2-H4folate revealed that
Lys-81 and Lys-352 were also involved in cross-linking with the
monoglutamate form. Mutants with single or multiple replacement(s) of
these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single
mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of
Lys-81 affected greatly the Km value for
5,10-CH2-H4PteGlu4 rather than for
5,10-CH2-H4folate. It is postulated that
Lys-352 may serve as the primary binding site to The glycine cleavage system is a multienzyme complex composed of
four proteins termed P-, H-, T-, and L-protein and catalyzes the
oxidative cleavage of glycine, a major catabolic pathway of glycine in
various organisms. T-protein is a folate-dependent enzyme
that catalyzes the formation of ammonia and
5,10-CH2-H4folate1
from the aminomethyl intermediate bound to the lipoate cofactor of
H-protein (reviewed in Ref. 1). T-protein should have the interaction
or binding site(s) with H-protein, the folate cofactor, and possibly
P-protein and the active site that directly participates in the
cleavage of the aminomethyl intermediate. While the kinetic properties
of the forward (2) and the reverse (3) reactions catalyzed by T-protein
have been extensively studied, little is known in terms of specific
amino acid residues of T-protein involved in binding or catalysis.
The primary structures of T-proteins from eight different species have
been determined so far (4-10). The sequence alignment by computer
program CLUSTAL W (11) indicates that there are relatively high
identities between T-proteins from plants and animals (46-88%), but
there are less identities between animal enzymes and the yeast and
Escherichia coli enzymes (~30%). Thus, the regions well
conserved among the eight species are very limited (see Fig.
1). A partial sequence homology of
T-proteins to -carboxyl group of
the first glutamate residue nearest the p-aminobenzoic acid
ring of 5,10-CH2-H4folate and
5,10-CH2-H4PteGlu4, whereas Lys-81
may play a key role to hold the second glutamate residue through
binding to
-carboxyl group of the second glutamate residue.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of corynebacterial sarcosine oxidase and rat
dimethylglycine dehydrogenase has been reported (12). The homologous
region was considered to be the tetrahydrofolate binding domain, since
all these enzymes catalyze the formation of
5,10-CH2-H4folate from different one-carbon donors (12). Physiological folate substrates generally have a string of
glutamate residues attached to the benzoyl moiety of the cofactor, and
the polyglutamylation enhances the cofactor binding to
folate-dependent enzymes (13-16). The interaction between the polyglutamate moiety and protein has been extensively investigated in thymidylate synthase (TS) that uses
5,10-CH2-H4folate as a substrate. Crystal
structure analysis revealed that TS binds the polyglutamate moiety with
a positively charged hydrophilic surface, where only the first
glutamate residue (Glu-1) nearest the p-aminobenzoic acid
ring is rigidly fixed. The lysine residue at the position 50 of
E. coli TS (the numbering of amino acid residues corresponds to that of Lactobacillus casei TS), which is nearly
invariant among all TS enzymes, participates in the binding to
-carboxyl group of Glu-1 (13).
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Fig. 1.
CLUSTAL W sequence alignment of T-proteins
from human (6), bovine (4), chicken (5), pea (8), potato (9),
Flaveria pringlei (9), Saccharomyces
cerevisiae (10), and E. coli (7) and
nomenclature of lysyl peptides obtained from ET. Identical
residues are shaded, and mutated residues are
boxed.
As part of an overall effort to identify the domains of T-protein
responsible for cofactor recognition, complex association, and
catalysis, we investigated specific lysine residues involved in the
binding with the glutamyl tail of folate derivatives. In the present
study, we utilized a carbodiimide-mediated covalent attachment of
14C-labeled
5,10-CH2-H4PteGlu4 to lysine
residues located at or near the folate-binding site of E. coli T-protein. Three lysine residues were identified, and their
role was confirmed by replacing these residues with glutamic acid by
site-directed mutagenesis.
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EXPERIMENTAL PROCEDURES |
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Materials--
[U-14C]glutamic acid (9.29 GBq/mmol) and NaH14CO3 (2.0 GBq/mmol) were
obtained from Amersham Pharmacia Biotech. Restriction endonucleases and
other DNA modifying enzymes were purchased from New England Biolabs,
Roche Molecular Biochemicals, Toyobo (Tokyo, Japan), or Takara Shuzo
(Shiga, Japan). Oligonucleotides were from Life Technologies, Inc.
Folic acid, lysylendopeptidase, and N-hydroxysuccinimide were purchased from Wako Pure Chemicals (Osaka, Japan) and EDC from
Sigma. Pteroyltetra--glutamate was obtained from Schircks Laboratories (Jona, Switzerland). V8 protease was obtained from Roche
Molecular Biochemicals. Folic acid and pteroyltetra-
-glutamate were
reduced (17) and used for synthesis of
5,10-CH2-H4folate and
5,10-CH2-H4PteGlu4, respectively,
as described (18).
Expression and Purification of Lactobacillus casei
Folylpolyglutamate Synthetase--
Plasmid pGT3-8.1, a pEMBL vector
carrying the L. casei FPGS chromosomal gene (19) kindly
supplied by Dr. A. L. Bognar of University of Toronto, was
transformed into E. coli MV1190. The bacteria were grown at
37 °C in 1000 ml of LB medium (20) containing 100 µg/ml
ampicillin. At the late log phase, the cells were harvested by
centrifugation (8,000 × g, 10 min), resuspended in 50 ml of 50 mM potassium phosphate buffer, pH 7.0, containing
50 mM KCl, and sonicated at 0 °C four times for 5 min
using a Branson Sonifier 250 with a 1/2-inch disrupter horn in the
pulse mode (output 8, 20% duty cycle). The sonicate was centrifuged at
105,000 × g for 60 min, and the supernatant was made
70% ammonium sulfate saturation. The precipitate was resuspended in 20 ml of buffer A (50 mM potassium phosphate, pH 6.8) and
extensively dialyzed against buffer A. The sample was applied to a
phosphocellulose column (P11, 2.5 × 8 cm, Whatman) equilibrated
with buffer A. The column was washed with 10 column volumes of buffer A
and eluted with a linear gradient of 0-500 mM KCl (600 ml)
in buffer A. The fractions containing the FPGS activity were pooled and
concentrated by 70% ammonium sulfate precipitation. The pellet was
resuspended in 3 ml of buffer B (50 mM potassium phosphate,
pH 7.0, containing 200 mM KCl), applied to a Sephacryl
S-100 column (2.6 × 90 cm, Amersham Pharmacia Biotech)
equilibrated with buffer B, and eluted with buffer B. Fractions
containing FPGS were concentrated with a Centriprep-10 concentrator
(Grace Japan, Tokyo, Japan) and stored at 20 °C.
FPGS activity was measured by the incorporation of [U-14C]glutamic acid into 5,10-CH2-H4folate essentially as described by Bognar and Shane (21). 0.12 mM 5,10-CH2-H4folate and 2 mM L-[U-14C]glutamic acid (20 MBq/mmol) were employed as substrates. A unit of FPGS activity represents 1 nmol of [U-14C]glutamic acid incorporated/min. The purified enzyme exhibited a specific activity of 660 units/mg.
Preparation of 14C-labeled
5,10-CH2-H4PteGlu4--
The
reaction mixture contained 100 mM Tris, 50 mM
glycine buffer, pH 9.3, 0.4 mM
5,10-CH2-H4folate, 4 mM
L-[U-14C]glutamic acid (195 MBq/mmol), 5 mM ATP, 10 mM MgCl2, 200 mM KCl, 5 mM DTT, 66.5 mM
formaldehyde, 0.01% bovine serum albumin, and 924 units of L. casei FPGS in a total volume of 4 ml. The mixture was incubated at
37 °C for 3 h, and then protein ingredients were removed by
filtration with a Centricon-10 concentrator (Grace Japan). The filtrate
was divided into three equal portions, and a portion was applied to a
reversed-phase C18 column (TSKgel ODS-120T, 4.6 × 250 mm, Tosoh, Tokyo, Japan) attached to a 655 HPLC system (Hitachi, Tokyo,
Japan). The column was developed with stepwise gradients of
acetonitrile using solution A (50 mM ammonium acetate, pH
7.1) and solution B (10% acetonitrile in solution A) at a flow rate of
1 ml/min. The absorbance of the effluent was monitored at 295 nm. The
collected peak fractions were immediately stored at 80 °C in small
aliquots until use. The samples were lyophilized to dryness with a
Speed Vac concentrator (Savant) and resolved in water. After the
determination of concentration, the material was immediately used for
cross-linking with ET. Concentrations of the folate derivative were
determined spectrophotometrically using
295 = 32.6 × 106 cm2/mol (22). The material was also
analyzed by scintillation counting, spectrophotometry, and
matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.
Expression and Purification of Recombinant E. coli
T-protein--
DNA manipulations were accomplished by standard
techniques (20). The pEGCV()5 plasmid containing the gcv
operon (7) was digested with EcoRV and self-ligated. The
product, pTZ/ETEH, contains gcvT and gcvH genes
and a part of gcvP gene. An NdeI site was
introduced adjacent to the codon for N-terminal Ala of gcvT
gene in pTZ/ETEH by oligonucleotide-directed mutagenesis with the sense
oligonucleotide
5'-TCAATGAGGACCATATGGCACAACAG-3' (the
NdeI site is underlined and modified bases are shown in
boldface letters) according to the method of Kunkel et al.
(23) using a Bio-Rad kit. Creation of a BamHI site placed 21 bases downstream of the stop codon and deletion of the NdeI
site originally present in the coding region without altering the amino
acid were simultaneously carried out with sense oligonucleotides,
5'-TTGGAGATTGATGGATCCGCAACGTACCAG-3' (the BamHI site is underlined and modified bases are shown
in boldface letters) and 5'-GATGTGTCACACATGACCATCGT-3' (the modified base is shown in a boldface letter), respectively. The resulted plasmid (pTZ/ETEHnb) was digested with NdeI and
BamHI, and the NdeI-BamHI fragment was
cloned into pET3a (24). The nucleotide sequence of the resultant
expression vector pET/ET was confirmed by nucleotide sequencing
employing a 373 DNA sequencing system (Perkin-Elmer).
E. coli BL21(DE3)pLysS cells (24) transformed with pET/ET
were grown in 200 ml of LB medium containing 20 µg/ml ampicillin and
30 µg/ml chloramphenicol. Expression was induced by the addition of
25 µM isopropyl--D-thiogalactopyranoside
at the start of the culture, and cells were incubated at 30 °C for
24 h.
Purification of recombinant ET was carried out as described for the
native ET (7) with some modifications. The cell-free extracts were
prepared with buffer C (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1 mM DTT, 10 µM
p-amidinophenylmethanesulfonyl fluoride) and subjected to
column chromatographies on DEAE-Sepharose CL-6B (2 × 6 cm,
Amersham Pharmacia Biotech), hydroxyapatite (2 × 6 cm), and
Sephacryl S-100 (2.6 × 90 cm). The elution from the
DEAE-Sepharose CL-6B column was performed with a linear gradient of
NaCl (0.1-0.3 M in 200 ml of buffer C). The hydroxyapatite
column was equilibrated with 5 mM potassium phosphate, pH
7.0, containing 0.5 mM DTT, and the elution was performed
by a linear gradient of potassium phosphate (5-100 mM, pH
7.0, in 200 ml) containing 0.5 mM DTT. The Sephacryl S-100
column was developed with 50 mM potassium phosphate, pH
7.0, containing 0.2 M NaCl. The final preparations were
pooled, dialyzed against 20 mM potassium phosphate, pH 7.0, concentrated with a Centriprep-10 concentrator and stored at 20 °C.
Purification of Recombinant E. coli P-protein--
Recombinant
E. coli P-protein was purified from E. coli
MV1190 cells transfected with pEGCV(+)5 (7) grown at 30 °C for 24 h in 1000 ml of LB medium containing 50 µg/ml ampicillin and 25 µM isopropyl--D-thiogalactopyranoside.
The purification was performed according to the method described
previously (7), except that the step with butyl-agarose column
chromatography was omitted. The final preparation was more than 90%
pure, as judged by SDS-PAGE. P-protein was assayed as described
previously (7).
Expression and Purification of E. coli H-protein--
For
expression of recombinant EH, gcvH gene with an
NdeI site adjacent to the codon for the N-terminal Ser
residue and a BamHI site 27 bases downstream from the stop
codon were amplified by polymerase chain reaction using pEGCV()5 (7)
as a template. The following pair of primers was used: a 5'-end primer,
5'-ACATATGAGCAACGTACCAGCAGAAC-3' (the
NdeI site is underlined and modified bases are shown in
boldface letters); a 3'-end primer,
5'-CGGATCCTCTCCCGCAGAAGAGG-3' (the
BamHI site is underlined and the modified base is shown in a
boldface letter). The product was purified with a Geneclean II kit (BIO
101) after agarose gel electrophoresis and digested with
NdeI and BamHI. The resultant DNA fragment was
cloned into pET3a. The nucleotide sequence of the expression vector,
pEH, was verified by DNA sequencing.
E. coli BL21(DE3)pLysS cells transfected with pEH were grown
in 800 ml of LB medium containing 20 µg/ml ampicillin, 30 µg/ml chloramphenicol, and 150 µM lipoic acid at 30 °C for
24 h. The expression of EH was induced by 25 µM
isopropyl--D-thiogalactopyranoside added at the start of
the incubation. Cell-free extracts were prepared as described
previously (7) with buffer D (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 10 µM
p-amidinophenylmethanesulfonyl fluoride) and heated in a
boiling water bath for 2 min. The supernatant of the heated sample was
subjected to column chromatographies on DEAE-Sepharose CL-6B (2.5 × 10 cm), hydroxyapatite (2 ×8 cm), and Sephacryl S-100 columns
(2.6 × 90 cm) essentially as described for the purification of
wild-type ET with minor modifications. The elution from the
DEAE-Sepharose column was performed with a linear gradient of NaCl
(0.28-0.45 M in 500 ml of buffer D). The buffer employed
in the hydroxyapatite column chromatography contained no DTT, and the
Centriprep-10 concentrator was replaced by a Centriprep-3 concentrator.
The mobility of EH in crude extracts and in the sample from each
purification step corresponded to that of lipoylated holoH-protein on
SDS-PAGE, indicating that the condition described above led to the full
lipoylation of overexpressed EH. The final preparation was more than
95% pure as judged by SDS-PAGE. The activity of H-protein was assayed
as described (7).
Location of Polyglutamate Binding Sites on E. coli T-protein-- Recombinant wild-type ET (20 nmol) was incubated for 15 min on ice with either 1.2 mM 5,10-CH2-H4folate or 0.3 mM 14C-labeled (3.2 × 104 dpm/nmol) or non-labeled 5,10-CH2-H4PteGlu4 in 50 mM HEPES buffer, pH 7.0, and then treated with 5 mM each of EDC and N-hydroxysuccinimide at 25 °C for 1 h. Wild-type ET alone was treated with the cross-linkers as a control. The reaction was quenched by adding 1.0 M Tris-HCl, pH 8.0, to the final concentration of 50 mM, and the reaction mixture was concentrated with a Microcon-10 concentrator (Grace Japan). The material was loaded onto a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, and eluted with the same solution. The resultant cross-linked products were carboxymethylated and digested with lysylendopeptidase as described previously (5). The digested mixture was applied to a reversed-phase C4 column (RP-304, 4.6 × 250 mm, Bio-Rad), and the column was eluted with four stage gradients of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min using a Hitachi 655 HPLC system. Absorbance was monitored at 220 nm. Radioactivity, when used, was monitored along with the absorbance at 220 nm. Peptides were analyzed by N-terminal sequencing using Perkin-Elmer 477A and Hewlett Packard G100A protein sequencers. When necessary, cross-linked peptides were digested with Staphylococcus aureus V8 protease (1/18-1/10, mol/mol) at 25 °C for 18 h in 100 µl of 50 mM ammonium bicarbonate, pH 7.8, 5% acetonitrile and separated by HPLC as described above but with a gradient of 0-48% acetonitrile in 0.1% trifluoroacetic acid.
Construction, Expression, and Purification of Mutant E. coli T-proteins-- Construction of some mutant ETs (K352E, K352Q, K352R, K75E, K81E, and K75E/K78E/K81E) was performed by in vitro mutagenesis according to the method of Kunkel et al. (23). For this purpose, pTZ/ETEHnb was trimmed by digestion with BamHI and self-ligated. The resulted plasmid was employed in the mutagenesis reaction with oligonucleotides listed in Table I. The NdeI-BamHI fragments were isolated from the mutant plasmids and ligated into pET3a. To construct K75E/K352E, K81E/K352E, or K75E/K78E/K81E/K352E mutant, each NdeI-Eco52I fragment (612 base pairs) from the plasmids carrying K75E, K81E, or K75E/K78E/K81E mutant and the Eco52I-BamHI fragment (510 base pairs) from K352E mutant plasmid were ligated in a one-step reaction into pET3a. Construction of K78E, K78E/K81E, K78E/K352E, or K78E/K81E/K352E mutant was performed by the megaprimer polymerase chain reaction mutagenesis procedure described by Sarkar and Sommer (25). For the first round of polymerase chain reaction, pET/ET was used as a template with mutagenic reverse primer in Table I and T7 promoter primer, 5'-TAATACGACTCACTATAGGG-3'. The double-strand polymerase chain reaction products were gel-purified and used as megaprimers in the second round of polymerase chain reaction along with T7 terminator primer, 5'-GCTAGTTATTGCTCAGCGGTG-3', using either pET/ET (for K78E and K78E/K81E) or the expression plasmid for K352E (for K78E/K352E and K78E/K81E/K352E) as a template. Amplified products were purified, digested with NdeI and BamHI, and ligated into pET3a. After verification of the mutations by DNA sequencing, the expression plasmids were transfected into E. coli BL21(DE3)pLysS cells. Expression and purification of mutant ETs were carried out as described for the recombinant wild-type ET.
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Kinetic Analysis-- Apparent Km values of mutant ETs for 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4 were determined at varied concentrations of either folate substrates with a constant concentration of other two substrates using the "coupled assay" (3) with some modifications. The reaction mixture contained in 0.25 ml: 50 mM Tris-HCl, pH 8.5, 10 mM DTT, 0.25 mM pyridoxal phosphate, 0.8 mM NADH, 20 mM NaH14CO3 (37 MBq/mmol), 1.2 mM formaldehyde, 50 units of E. coli P-protein, 32 µg of diaphorase (pig heart, Roche Molecular Biochemicals), 6 µM reduced EH, 5,10-CH2-H4folate or 5,10-CH2-H4PteGlu4, 200 mM NH4Cl, and T-protein. The reaction was initiated by the addition of reduced EH, continued for 3 min at 37 °C, and terminated by the addition of 12.5 µl of 50% perchloric acid. The mixture was bubbled with CO2 and the remaining radioactivity was determined by scintillation counting. Concentrations of 5,10-CH2-H4folate were 40-360 µM for the experiments with wild-type ET, K352E, K352Q, K352R, K75E, K78E, K81E, and K78E/K352E and 0.133-1.2 mM with K75E/K352E, K81E/K352E, K78E/K81E, K78E/K81E/K352E, K75E/K78E/K81E, and K75E/K78E/K81E/K352E. Concentrations of 5,10-CH2-H4PteGlu4 were 4-36 µM for the experiments with wild-type ET, K352E, K352Q, K352R, and K78E, 20-180 µM with K75E, K81E, and K78E/K352E, 67-600 µM with K78E/K81E, 0.2-1.8 mM with K75E/K352E and K75E/K78E/K81E, and 0.4-3.6 mM with K81E/K352E, K78E/K81E/K352E, and K75E/K78E/K81E/K352E. Mutant ETs of 20 ng was employed in the experiments with 5,10-CH2-H4folate. With 5,10-CH2-H4PteGlu4, 25 ng of mutant ETs except that 15 ng of K75E/K352E, K75E/K78E/K81E, K81E/K352E, and K78E/K82E/K352E was employed.
Mass Spectrometry--
The mass spectroscopic analysis of
folylpolyglutamates separated by a reversed-phase
C18-column was performed by matrix-assisted laser
desorption ionization/time-of-flight mass spectrometry using a Voyager
RP mass spectrometer (PerSeptive Biosystems). The samples containing
~3.6 pmol of folate were employed and 1% -cyano-4-hydroxycinnamic acid in 33% acetonitrile containing 0.1% trifluoroacetic acid was
employed as a matrix. Des-Arg1-bradykinin (904.04 + 1 Da)
and potassium ion (39.00 Da) were used as external standards.
Other Methods--
Protein concentration was routinely
determined by the method of Bradford (26) with bovine serum albumin as
a standard. Concentrations of purified wild-type and mutant ETs and EH
were also estimated by amino acid analysis using a Hitachi 830 amino
acid analyzer. Comparison of the data obtained by Bradford method and
the amino acid analysis revealed that the amounts of proteins estimated with Bradford method were somewhat greater than those obtained by amino
acid analysis. Concentrations of ET and EH can be calculated from the
data obtained by Bradford method with multiplying 0.7 for ET and 0.9 for EH. SDS-PAGE was carried out by the method of Laemmli (27).
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RESULTS |
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Preparation of 14C-labeled
5,10-CH2-H4PteGlu4--
14C-Labeled
5,10-CH2-H4PteGlu4was enzymatically
synthesized from 5,10-CH2-H4folate and
L-[U-14C]glutamic acid using purified
recombinant L. casei FPGS, and the products were separated
by HPLC on a reversed-phase C18 column. Formaldehyde was
added to the reaction mixture to prevent the release of
methylene-carbon from the folate substrate during incubation. The
products and substrate were well separated by stepwise gradients elution of acetonitrile in 50 mM ammonium acetate, pH 7.1 (Fig. 2). Four peak fraction showing
characteristic absorption spectra of
5,10-CH2-H4folate with maximum at around 295 nm
were obtained, and their properties were summarized in Table
II. Molecular masses of P1 and P2 were in
good agreement with that of
5,10-CH2-H4PteGlu4 (844.298 + 1 Da), whereas those of P3 and P4 were consistent with that of
5,10-CH2-H4folate (457.171 + 1 Da). These
results agree well with the amounts of the incorporated radioactive
glutamate residues.
5,10-CH2-H4PteGlu4 prepared from
chemically synthesized pteroyltetra--glutamate gave the same
retention time as P2 (data not shown). P1 and P2 gave the same
cross-linked lysylpeptides when used to cross-linking with ET. In
addition, both P1 and P2 served as a substrate for T-protein. In the
following cross-linking experiments, P2 fraction was employed as
14C-labeled
5,10-CH2-H4PteGlu4.
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Localization of the Folate Binding Sites by Cross-linking and
Lysylpeptide Mapping--
Wild-type ET was preincubated with
14C-labeled
5,10-CH2-H4PteGlu4 and then
cross-linked using EDC and N-hydroxysuccinimide. The ratio
of covalently attached
5,10-CH2-H4PteGlu4/mol of ET was
near unity. The cross-linked product was digested with
lysylendopeptidase and the resultant peptides were separated by HPLC
(Fig. 3B). As a control, ET
alone was treated with the cross-linkers and subjected to lysylpeptide
mapping (Fig. 3A) and Edman sequencing. The peaks P1, P2,
and P4 were radioactive and not observed in the control experiment. The
another radioactive peak, P3, was inseparable from peak P', and the
absorbance at 220 nm was greater than that of P' (Fig. 3, A
and B). The initial unnamed radioactive peak (Fig.
3B) did not represent peptide(s) cross-linked with
5,10-CH2-H4PteGlu4 since the
radioactivity could not be retained on the sequencing column of the
Hewlett Packard G100A protein sequencer. The four peaks (P1-P4) were
isolated in the parallel experiment with the non-labeled folate and
subjected to Edman sequencing. As shown in Table
III, P1 and P2 gave the sequences
corresponding to residues 76-81 with Lys-78 missing and residues
350-360 with Lys-352 missing, respectively. P4 gave the sequence
starting from residue 79 with Lys-81 missing. In the control
experiment, these peptides were cleaved at the C-terminal side of the
lysine residues not detected in the above Edman degradation. Namely,
peptide P1 was cleaved to peptides 76-78 (K2, Fig. 1) and 79-81 (K3,
Fig. 1), peptide P2 to peptides 350-352 (K12, Fig. 1) and 353-360
(K13, Fig. 1), and peptide P4 to peptides 79-81 and 82-119 (K4, Fig.
1). The cleavage of P2 was incomplete due to the resistance of the
lysyl-proline bond at 352-353 to lysylendopeptidase (Fig.
3A). K2 and K3 were eluted from the C18
column in the void volume fraction and K4 was eluted next to P4 (Fig.
3, A and B). These results indicate that the
above three lysine residues could not be detected in the Edman
degradation due to the modification and, therefore, are likely the
candidates for the cross-linking residues with the polyglutamate moiety
of 5,10-CH2-H4PteGlu4.
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Peak P' (Fig. 3A) contained peptides K1, K5, and K7. N-terminal sequencing of P3 up to 20 residues showed the existence of peptide K6-7 whose Lys-157 was not modified, in addition to the three peptides mentioned. P3 was digested with S. aureus V8 protease under the specific condition for glutamic acid C-terminal cleavage, and the peptides were separated by HPLC. No radioactive peak was detected and no evidence for the modification of amino acid residues was obtained by amino acid sequencing of all the V8 peptides (data not shown). Peptide K6-7 with intact Lys-157 was not yielded from the control experiment and from the ET cross-linked with 5,10-CH2-H4folate. Therefore, the inability of lysylendopeptidase to cleave the C-terminal side of Lys-157 may be attributed to some kind of interaction between Lys-157 and 5,10-CH2-H4PteGlu4.
The parallel cross-linking experiment with 5,10-CH2-H4folate gave cross-linked peptide peaks P5 and P6 (Fig. 3C), which had the same amino acid sequence as P2 and P4, respectively. The results indicate that Lys-352 and Lys-81 were also involved in the coupling with the carboxyl group(s) of the monoglutamate residue. Peptide peaks corresponding to P1 and P3 were not detected (Fig. 3C).
ET with covalently cross-linked 5,10-CH2-H4folate has no significant enzymatic activity with or without externally added folate substrate.
Effects of the Mutations of the Folate Binding Sites-- To assess the contribution for interaction with folate substrate, above three lysine residues (Lys-78, Lys-81, and Lys-352) and Lys-75 were subjected to site-directed mutagenesis. Lys-352, the residue absolutely conserved among T-proteins from eight various species (Fig. 1), was mutated to glutamic acid, glutamine, or arginine. Other lysine residues were replaced with glutamic acid. In addition to the replacement of a single residue, multiple replacements with glutamic acid were carried out. All mutant proteins were highly expressed in E. coli BL21(DE3)pLysS cells as soluble protein and purified by the same strategy used for the wild-type enzyme. The purified proteins gave a single band on SDS-PAGE (not shown). Apparent Km values of mutant ETs for 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4 were determined and summarized in Table IV. Wild-type ET showed 6.5-fold higher affinity for 5,10-CH2-H4PteGlu4 than that for 5,10-CH2-H4folate. The single mutation of Lys-352 to Glu and Gln increased Km values for both folate substrates about 2-fold, whereas mutation to Arg caused no effect. Thus, the side chain positive charge appears to be essential for the interaction. Mutation of Lys-78 to Glu increased Km values for both folate substrates 1.4-fold. The K81E mutant showed a 3-fold increase in Km for 5,10-CH2-H4folate and a 16-fold increase in Km for 5,10-CH2-H4PteGlu4. The contribution of Lys-75 was not evidenced by the cross-lining experiment but the mutation of Lys-75 to Glu increased Km for 5,10-CH2-H4folate 2.5-fold and Km for 5,10-CH2-H4PteGlu4 8-fold. The multiple mutations resulted in a 7-13-fold increase in Km values for 5,10-CH2-H4folate and a 5-252-fold increase in Km values for 5,10-CH2-H4PteGlu4. Mutation of above lysine residues affected slightly the affinity of ET for the protein substrate EH. Km values for EH of wild-type ET and the mutant K75E/K78E/K81E/K353E were 0.45 µM and 0.53 µM, respectively. Changes in kcat values were not so significant, and even higher values were obtained (Table IV).
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DISCUSSION |
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In this study, 14C-labeled 5,10-CH2-H4PteGlu4 was used to investigate the folate binding sites on E. coli T-protein. 5,10-CH2-H4PteGlu4 whose terminal three glutamate residues were uniformly labeled with 14C was enzymatically synthesized from 5,10-CH2-H4folate and [U-14C] glutamic acid using L. casei FPGS. The L. casei enzyme was chosen because of its preference for 5,10-CH2-H4folate as the substrate among various folates (21). The radiolabeled products separated by HPLC were solely tetraglutamate derivatives, and no other polyglutamate forms were obtained under the conditions used here (Table II). The products were separated into two peak fractions by HPLC (P1 and P2, Fig. 2), but they behaved similarly as a substrate for ET and gave the same cross-linked lysylpeptides when cross-linked with ET. The differences of these two fractions were not investigated further.
EDC was employed to produce zero-length cross-linking between contacted
carboxyl groups of polyglutamate moiety of
5,10-CH2-H4PteGlu4 and amino groups
of ET in the binary complex. Binding of EDC-activated folate
derivatives to folate-dependent enzymes has been previously reported with thymidylate synthase (28) and
5,10-methenyltetrahydrofolate synthetase (14). In a preliminary
experiment, we activated
5,10-CH2-H4PteGlu[14C]Glu3
with EDC and N-hydroxysuccinimide prior to interact with ET
under the widely used conditions (29). No significant amounts of
labeled peptides were obtained after lysylendopeptidase digestion indicating that the condition employed was not appropriate for the
cross-linking. Considerable amounts of cross-linked product were
obtained when ET and the folate substrate were mixed first to
facilitate the complex formation and then EDC and
N-hydroxysuccinimide were introduced to the mixture. The
Edman sequencing of covalently radiolabeled lysylpeptides revealed that
three lysine residues (Lys-78, Lys-81, and Lys-352) were involved in
cross-linking with polyglutamate moiety of
5,10-CH2-H4PteGlu4. Two of them,
Lys-81 and Lys-352, were also implicated in the cross-linking with
5,10-CH2-H4folate. The -carboxylic acid and
-carboxylic acid groups of 5,10-CH2-H4folate were apparently involved in the contact with these two lysine residues.
The contribution of these lysine residues to the binding of the
folate substrates with ET was confirmed by site-directed mutagenesis and kinetic analysis. Kinetic analyses were conducted employing saturated amounts of H-, P-, and L-protein (diaphorase).
5,10-CH2-H4PteGlu4 was better
substrate for wild-type ET than
5,10-CH2-H4folate (Table IV), in good agreement
with other folate-dependent enzymes (13-16). Mutation of
Lys-352 or Lys-78 to glutamate increased Km values
for both folate substrates 2-fold or less, whereas mutation of Lys-81
to glutamate affected greatly the Km value for
5,10-CH2-H4PteGlu4 (16-fold) than
for 5,10-CH2-H4folate (3-fold) (Table IV). The
results were consistent with the observations about the interaction of
folate derivatives with 5,10-methenyltetrahydrofolate synthetase. Maras
et al. (14) reported that Lys-18 of rabbit liver
5,10-methenyltetrahydrofolate synthetase interacts with the
-carboxyl of the Glu-1 residue of folate derivatives.
Km values for 5,10-methenyltetrahydrofolate and
5,10-methenyltetrahydropteroylpentaglutamate are 2.6- and 105-fold,
respectively, less than that for 5,10-methenyltetrahydropteroate, indicating that interaction of Glu-1 with the enzyme affected not so
greatly on the Km value and positively charged amino
acid residues other than Lys-18 may be important for the binding of the
polyglutamate form. The double mutant K81E/K352E showed a greater
increase in the Km value for
5,10-CH2-H4PteGlu4 (99-fold) than
for 5,10-CH2-H4folate (8-fold). These results
indicate that the interaction with Lys-81 of
5,10-CH2-H4PteGlu4 is more critical
than that of 5,10-CH2-H4folate for the binding
to ET. Based on the crystal structure of E. coli TS, Kamb
et al. (13) reported that glutamate residues of
5,10-CH2-H4PteGlu4 interact with a
positively charged hydrophilic surface of the protein where Glu-1 is
rigidly fixed through its
-carboxyl group with Lys-50, a nearly
invariant residue among all TS enzymes. Lys-352 of ET is conserved
among T-proteins so far studied, whereas Lys-78 and Lys-81 are not
(Fig. 1). Considering these circumstances, it is tempting to postulate
that Lys-352 interacts with the
-carboxyl group of Glu-1 of both
folate substrates and Lys-81 interacts with the
-carboxyl group of
the second glutamate residue (Glu-2) of
5,10-CH2-H4PteGlu4 and the
-carboxyl group of Glu-1 of
5,10-CH2-H4folate. The similarity in
Km values of K81E for both folate substrates may
reflect the impaired binding ability to Glu-2 of
5,10-CH2-H4PteGlu4.
As mentioned above, negatively charged glutamate residues of folylpolyglutamate lie in a position where they can interact with surface region of E. coli TS having positive potential (13). Maras et al. (14) suggested that a region containing multiple Lys and Arg residues serves as a polyglutamate binding site of rabbit liver 5,10-methenyltetrahydrofolate synthetase. Therefore, we investigated the role of Lys-75 situated in the vicinity of Lys-81 and Lys-78 of ET. Although cross-linking of folate derivatives with Lys-75 could not be demonstrated, K75E mutant showed significant increases in Km values for both folate substrates especially for 5,10-CH2-H4PteGlu4. The result together with the changes in Km values for folate substrates of mutants containing K78E mutation suggests that Lys-75 and Lys-78 are implicated in interaction with polyglutamate tail of 5,10-CH2-H4PteGlu4.
Multisites mutations with substitutions of both Lys-81 and Lys-352 gave higher Km values for 5,10-CH2-H4PteGlu4 than for 5,10-CH2-H4folate. kcat/Km values of these mutants with 5,10-CH2-H4PteGlu4 as substrates are 26-120-fold lower than that of wild-type ET (Table IV). The mutation probably induced electrostatic repulsive effects on the binding of folate substrates. Increase in kcat values of mutant ETs is presumably the results of the repulsive effects that enhance the release of the folate reaction products and accelerate the turnover of ET.
Chlumsky et al. (12) reported that an ~380-residue region
near the C terminus of subunit of Corynebacterium sp.
sarcosine oxidase exhibits homology with the C-terminal half of rat
dimethylglycine dehydrogenase and a region in the middle of T-protein.
Because these proteins all catalyze the synthesis of
5,10-CH2-H4folate from tetrahydrofolate and
various one-carbon donors, they suggested that these regions were
tetrahydrofolate binding domains. The above mentioned lysyl residues of
ET, however, are not situated in the region of T-protein they indicated.
Lys-75, -78, and -81 are not conserved in other T-proteins; therefore,
the putative folate-binding site discussed in this study seems to be
specific for ET, and there must be distinct folate-binding sites in
T-proteins from other species. In this connection, it is worth noting
that there is considerable variability in the region of TS that
contacts the second and third glutamate residues of polyglutamate tail
(13). Details of interaction of T-protein with the folate substrates
require further elucidation; crystal structure analysis is essential.
However, the present cross-linking study provides the first evidence of
the folate binding sites on T-protein.
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ACKNOWLEDGEMENTS |
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We thank Dr. Andrew L. Bogner of University of Toronto for the generous gift of L. casei FPGS clone, Dr. Hiroshi Munakata of Kinki University and Ryoichi Kunai of the University of Tokushima for help in amino acid sequence analysis, and Dr. Shiro Futaki of Kyoto University and Yasuko Yoshioka of the University of Tokushima for help in mass spectrometry.
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FOOTNOTES |
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* This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Inst. for Enzyme
Research, University of Tokushima, Kuramotocho 3-chome, Tokushima 770-8503, Japan. Fax: 81-886-33-7428.
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ABBREVIATIONS |
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The abbreviations used are: 5,10-CH2-H4folate, methylenetetrahydrofolate; TS, thymidylate synthase; 5,10-CH2-H4PteGlu4, methylenetetrahydropteroyltetraglutamate; DTT, dithiothreitol; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EH, E. coli H-protein; ET, E. coli T-protein; FPGS, folylpolyglutamate synthetase; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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