From the Department of Microbiology and Molecular
Genetics, University of Medicine and Dentistry of New Jersey
(UMDNJ)-New Jersey Medical School, International Center for
Public Health, Newark, New Jersey 07101, § Institute of
Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Pozna
,
Poland, and
Department of Biochemistry and Biotechnology,
Agricultural University, 60-637 Pozna
, Poland
Received for publication, November 20, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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Editing of the amino acid
homocysteine (Hcy) by certain aminoacyl-tRNA synthetases results
in the formation of an intramolecular thioester, Hcy-thiolactone. Here
we show that the plant yellow lupin, Lupinus luteus, has
the ability to synthesize Hcy-thiolactone. The inhibition of
methylation of Hcy to methionine by the anitifolate drug aminopterin
results in greatly enhanced synthesis of Hcy-thiolactone by L. luteus plants. Methionine inhibits the synthesis of
Hcy-thiolactone in L. luteus, suggesting involvement of
methionyl-tRNA synthetase. Consistent with this suggestion is our
finding that the plant Oryza sativa methionyl-tRNA
synthetase, expressed in Escherichia coli, catalyzes
conversion of Hcy to Hcy-thiolactone. We also show that Hcy is a
component of L. luteus proteins, most likely due to facile
reaction of Hcy-thiolactone with protein amino groups. In
addition, L. luteus possesses constitutively expressed,
highly specific Hcy-thiolactone-hydrolyzing enzyme. Thus,
Hcy-thiolactone and Hcy bound to protein by an amide (or peptide)
linkage (Hcy-N-protein) are significant components of plant
Hcy metabolism.
Homocysteine
(Hcy)1-thiolactone, a cyclic
thioester of Hcy, was discovered by serendipity almost 70 years ago as
a by-product of the digestion of methionine with hydriodic acid, a
procedure used then for the determination of protein methionine (1). The discovery of an error editing reaction of aminoacyl-tRNA
synthetases, in which Hcy is converted to Hcy-thiolactone,
highlighted the biological significance of Hcy-thiolactone (2).
Hcy-thiolactone is synthesized by methionyl-tRNA synthetase (MetRS) in
bacterial (3-6), yeast (6-8), and mammalian, including human, cells
(9-17). Isoleucyl-tRNA synthase and leucyl-tRNA synthase, in
addition to MetRS, synthesize Hcy-thiolactone from exogenous Hcy, at
least in bacteria (5). Hcy-thiolactone forms in a two-step reaction driven by the hydrolysis of ATP (2). In the first step, MetRS catalyzes
reaction of Hcy with ATP, which leads to the formation of a
MetRS-bound homocysteinyl adenylate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
In the second step, MetRS catalyzes the reaction of the side chain
thiolate of Hcy, which displaces the AMP moiety from the activated
carboxyl group of Hcy; Hcy-thiolactone is a product of this reaction
(Reaction 2).
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Reaction 2.
Hcy-thiolactone, an intramolecular thioester of Hcy, is
relatively stable and has a half-life of about 25 h under
physiological conditions of pH and temperature (10). Because the
pK of its amino group is unusually low at 7.1 (18),
Hcy-thiolactone freely diffuses through cellular membranes and
accumulates in extracellular fluids (3-17). A characteristic
ultraviolet absorption spectrum with a maximum at 240 nm allows facile
detection and quantification of Hcy-thiolactone in biological samples
(4, 5, 19). Like all thioesters, Hcy-thiolactone is chemically
reactive. For example, Hcy-thiolactone forms adducts with protein
(Hcy-N-protein), in which the carboxyl group of Hcy is
linked by an amide bond with -amino group of a protein lysine
residue (10, 15, 20). The modification by Hcy-thiolactone results in
protein damage (12-17, 20).
Although its role in cell physiology is largely unknown, Hcy-thiolactone has been suggested to be a positive effector of the stationary phase response in Escherichia coli (21) and is also likely to be involved in the regulation of methionine synthase gene expression in E. coli (6).
In humans, Hcy-thiolactone is likely to play a role in cardiovascular disease due to its ability to form Hcy-N-protein, which leads to protein damage (10-17, 20). A protein component of high-density lipoproteins, Hcy-thiolactonase/paraoxonase, detoxifies Hcy-thiolactone, thereby minimizing formation of Hcy-N-protein in humans (22, 23).
Whether Hcy-thiolactone is present and how it is metabolized in plants
was unknown. Here we report that Hcy-thiolactone and Hcy-N-protein are components of Hcy metabolism in yellow
lupine (Lupinus luteus). We also show that Hcy-thiolactone
is synthesized by rice (Oryza sativa) methionyl-tRNA
synthetase and degraded by a highly specific yellow lupine
Hcy-thiolactone hydrolase.
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MATERIALS AND METHODS |
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[35S]Sulfur Amino Acids-- Carrier-free L-[35S]Met, from Amersham Biosciences, was supplemented with unlabeled methionine (Sigma- Aldrich) to a specific activity of 20,000 Ci/mol. L-[35S]Hcy-thiolactone (20,000 Ci/mol) was prepared by digestion of L-[35S]Met with hydriodic acid (1) as described previously (24). L-[35S]Hcy was prepared by hydrolysis of L-[35S]Hcy-thiolactone with 0.1 M NaOH at 37 °C for 15 min. The preparations of [35S]Met and [35S]Hcy were confirmed to be free of Hcy-thiolactone (<0.1%).
Lupine Seed Germination and 35S Labeling
Conditions--
Yellow lupine (L. luteus, var. Juno) seeds
were germinated at 21 °C on cellulose paper towels soaked with
deionized sterile water. On the 6th day, the roots were
removed, and the seedlings were transferred into 50-ml Falcon tubes (5 seedlings/tube) containing 1 ml of sterile water or 25 µM
aminopterin (Sigma-Aldrich) in water. Hypocotyls tips were immersed in
the liquid medium. After 48 h, the seedlings were transferred for
12 h to fresh tubes containing 1.5 µM
[35S]Met or [35S]Hcy (15 µCi in 0.5 ml of
sterile water). 35S-amino acids were used as tracers to
facilitate monitoring of Hcy-thiolactone during purification;
quantification was by measurements of A240 (see
below). Antifolate drugs, aminopterin, sulfonamide, or trimethoprim
(from Sigma-Aldrich), were included as indicated under "Results."
The hypocotyls and coltyledons were then collected separately and
frozen at 20 °C.
Preparation of L. luteus Extracts-- Yellow lupine hypocotyls or cotyledons (~1 g) were ground up at 0 °C with 1 ml of 50 mM potassium phosphate buffer, pH 7.5, using a mortar and pestle. The extracts were centrifuged at 30,000 × g using a JA25.50 rotor in a Beckman-Coulter J2 centrifuge (15 min, 2 °C).
Determination of Hcy-thiolactone in L. luteus-- The plant yellow lupine extracts (0.5-1 ml) were adjusted to pH 8 with di-potassium phosphate, and Hcy-thiolactone was extracted with 4 volumes of chloroform-methanol (2:1, v/v). Organic layers were reextracted with 0.1 M HCl. Aqueous layers were lyophylized on a Labconco concentrator and taken up in 50 µl of water, and 5-µl aliquots were subjected to two-dimensional TLC on 6.7 × 5-cm cellulose plates (Analtech, Newark, DE), as described previously (26). The first dimension separation was for 30 min using 1-butanol/acetic acid/water (4:1:1, v/v). The second dimension was for 15 min using isopropanol/ethyl acetate/water/ammonia (20:20:5:0.05, v/v). [35S]Hcy-thiolactone spots were visualized by autoradiography using Kodak BioMax x-ray film, scraped off the TLC plates, extracted with 60 µl of water, and finally quantified by cation exchange HPLC (19).
Measurements of Hcy-thiolactone Synthesis by Rice (O. sativa)
MetRS--
Plasmid pET/MOsC, encoding rice MetRS (25), was kindly
supplied by Marc Mirande (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France). The plasmid was introduced into competent E. coli BL21 (DE3) host cells (Promega). The cells were
propagated on M9 minimal medium supplemented with 1 µg/ml thiamine
and 50 µg/ml kanamycin (Sigma-Aldrich). Fresh overnight cultures were diluted 5-fold into fresh medium and grown for 2 h at 37 °C.
The cultures were then shifted to 30 °C, supplemented with 0.2 mM Ile, 0.2 mM Leu, 0 or 1 mM
isopropyl-
-D-thiogalactopyranoside, and 2.5 mM D,L-Hcy. Samples of cultures were analyzed
for Hcy-thiolactone by cation exchange HPLC (19).
Determination of Hcy-N-protein-- Proteins, extracted from hypocotyls of 6-day-old yellow lupine seedlings, were treated with 5 mM DTT for 5 min at room temperature and precipitated with 80% ethanol at 0 °C to remove free Hcy. The plant protein was dissolved in phosphate-buffered saline containing 5 mM DTT and precipitated with 80% ethanol. The cycle of DTT treatment and ethanol precipitation was repeated four more times. This procedure removed >99% total Hcy from plant protein extracts. Samples of DTT-treated protein were diluted to 0.1 ml with 25 mM DTT and transferred to glass ampoules (1-ml volume) containing 0.1 ml of 12 N HCl. The ampoules were sealed under vacuum, and the samples were hydrolyzed at 120 °C for 1 h. This procedure quantitatively converted Hcy-N-protein into Hcy-thiolactone. After hydrolysis, samples were lyophilized and dissolved in 10 µl of water, and 3.3-µl aliquots were subjected to two-dimensional TLC on 6.7 × 5-cm cellulose plates (Analtech) as described previously (26). [35S]Hcy-thiolactone, localized on TLC plates by autoradiography using Kodak BioMax x-ray film, was extracted with water (60 µl) and finally purified and quantified by cation exchange HPLC (19, 26). To determine Hcy-N-protein relative to protein methionine in lupine plants, radiolabeled spots corresponding to Hcy-thiolactone and methionine were cut out from a duplicate set of TLC separations and quantified by using a Beckman LS6500 scintillation counter.
Determination of Total Hcy-- The principle of the procedure involves the conversion of Hcy to Hcy-thiolactone, which is then quantified by HPLC (19). Plant extracts were treated with 5 mM DTT to convert disulfide-bound forms of Hcy to free reduced Hcy, deproteinized by ultrafiltration through Millipore 10-kDa cut-off membranes at 4 °C. The ultra-filtrate (50 µl) was lyophilized on a SpeedVac concentrator and dissolved in 6 µl of 50 mM DTT, and Hcy was converted to Hcy-thiolactone by treatment with 3 µl of 6 M HCl for 30 min at 100 °C. After lyophylization, samples were dissolved in 50 µl of water and subjected to HPLC.
HPLC Chromatography-- HPLC analyses were carried out using a cation exchange PolySULFOETHYL Aspartamide column (2.1 × 200 mm, 5 µ, 300 Å) from PolyLC, Inc. and System Gold Noveau HPLC instrumentation from Beckman-Coulter as described previously (19, 26). Solution A (10 mM mono-sodium phosphate) and solution B (200 mM NaCl in 10 mM mono-sodium phosphate) were used as solvents. After application of sample, the column was eluted with a linear gradient from 50% to 100% solution B for 5 min, followed by 100% solution B for 0.5 min and 2-min reequilibration with 50% solution B.
The effluent was monitored at multiple wavelengths, including
A240, the UV absorption maximum of
Hcy-thiolactone ( = 3,500 M
1
cm
1) (5, 15). For each sample, the identity of the eluted
material as Hcy-thiolactone was confirmed by its co-migration with an
authentic Hcy-thiolactone, by its characteristic absorbance spectrum
with a maximum at A240, and by its sensitivity
to lupine Hcy-thiolactonase or NaOH. The detection limit was 5 pmol of
Hcy-thiolactone.
Enzyme Assays-- Unless indicated otherwise, incubations were carried out at 37 °C in 0.1 M potassium-Hepes buffer (pH 7.2). Hcy-thiolactonase activity was determined by following the formation of [35S]Hcy from [35S]Hcy-thiolactone. Hcy was separated from Hcy-thiolactone by TLC on cellulose plates (Analtech) and quantified by scintillation counting (22, 23).
Spectrophotometric assays were used in substrate specificity studies
with nonradiolabeled substrates (all from Sigma). Hydrolysis of
Hcy-thiolactones was determined from the decrease of their characteristic UV absorption at = 240 nM (
= 3,500 M
1 cm
1) (22).
Hydrolysis of phenyl acetate and p-nitrophenyl acetate was
determined spectrophotometrically using
= 1,300 M
1 cm
1 at 270 nm for phenol and
= 13,000 M
1 cm
1 at 412 nm for p-nitrophenol, respectively. Hydrolysis of diethyl p-nitrophenyl phosphate (paraoxon) was measured
spectrophotometrically using
= 13,000 M
1 cm
1 at 412 nm for
p-nitrophenol (22).
In experiments in which utilization of other (thio)esters (10 mM) by Hcy-thiolactonase was tested, potential substrates and products were separated by TLC and visualized by staining with ninhydrin or under UV. With all potential substrate-product pairs, complete separation was achieved on cellulose plates (Analtech) using 1-butanol/acetic acid/water (4:1:1, v/v) as a solvent. Complete separation of acetyl-S-CoA (Sigma) and Met-S-CoA (prepared as described in Ref. 27) thioesters from free CoA-SH was achieved on polyethyleneimine-cellulose plates (Sigma) using 1.2 M LiCl as a solvent.
Purification of Hcy-thiolactone Hydrolase-- All steps were carried out at 4 °C. Buffer A, B, or C containing 10, 20, or 50 mM potassium phosphate (pH 6.8), respectively, 1 mM 2-mercaptoethanol, and 5% glycerol was used.
Yellow lupine seed (L. luteus, var. Juno) meal (100 g) was
extracted with 300 ml of Buffer A. Protein (9,030 mg) in crude extract,
obtained by centrifugation at 20,000 × g for 30 min, was fractionated with ammonium sulfate. Protein (2,320 mg)
precipitated between 0-35% ammonium sulfate saturation was collected
by centrifugation, dissolved in 5 ml of Buffer B, and extensively
dialyzed against Buffer B. Dialysate was clarified by centrifugation
and applied on DEAE-Sephacel column. The column was washed with 5 volumes of Buffer B and eluted with a KCl gradient in Buffer B. Protein fractions with Hcy-thiolactonase activity (60 mg), eluting at 0.3-0.35
M KCl, were concentrated by ammonium sulfate precipitation, dissolved in 5 ml of Buffer C, and further purified by gel filtration on a Superdex 200 column equilibrated with Buffer C. The enzyme eluted
from the gel filtration column as a protein with a molecular mass of 55 kDa. Active fractions (0.7 mg of protein) were
applied on a hydroxylapatite column equilibrated with Buffer A and
eluted with a gradient 10-100 mM phosphate, pH 6.8, in
Buffer A. Active fractions, eluted at 75 mM phosphate, were
concentrated, dialyzed against Buffer A, and stored at 20 °C. The
Hcy-thiolactonase preparation (0.2 mg of protein) had a specific
activity of 536 µmol/mg/h and was purified 25,000-fold. The purified
enzyme preparation showed several protein bands on SDS-PAGE.
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RESULTS |
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Synthesis of Hcy-thiolactone in Yellow Lupine Seedlings Increases
upon Depletion of Tetrahydrofolate--
In plants, Hcy is synthesized
de novo from sulfate and also as a by-product of cellular
methylation reactions (Fig. 1) (28, 29).
Three pathways of further Hcy metabolism are utilized to different
extents by living organisms: methylation to methionine, trans-sulfuration to cysteine, and conversion to Hcy-thiolactone. In
plants, Hcy is further metabolized by methylation to methionine by a
methyltetrahydofolate-dependent methionine synthase (Fig. 1,
MS) (29) or by
S-methyl-methionine-dependent Hcy
S-methyltransferase (29-31). Trans-sulfuration of Hcy to
cysteine, present in fungi and mammals, is absent in plants (29).
MetRS-dependent metabolism of Hcy to Hcy-thiolactone,
present in bacteria, yeast, and mammalian cells (15-17), was not known
to be present in plants.
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To determine whether metabolism of Hcy to Hcy-thiolactone occurs in
plants, yellow lupine seedlings were examined for the presence of
Hcy-thiolactone and Hcy-thiolactone hydrolase. Before extraction of
Hcy-thiolactone, 6-day-old seedlings were maintained for additional
60 h on water in the absence and presence of the antifolate drug
aminopterin (25 µM), an inhibitor of eukaryotic dihydrofolate reductase enzymes (32). To facilitate purification of
Hcy-thiolactone from plant tissues, yellow lupine seedlings were
metabolically labeled with radiotracers [35S]Met or
[35S]Hcy for 12 h before harvesting. As shown in
Figs. 2B and 3, Hcy-thiolactone was present in hypocotyls of yellow lupine seedlings. Although exogenous 35Samino acids were taken up by
seedlings and metabolized to [35S]Hcy-thiolactone
intracellularly (Fig. 2B), their contribution to total
Hcy-thiolactone, measured by A240 (Fig.
3) synthesis, was <0.1%. Lupine tissue
concentrations of Hcy-thiolactone were 49.5 µM and <0.6
µM in the presence and absence of aminopterin, respectively (Table I). The presence of
up to 1 mM exogenous [35S]Hcy in culture
medium did not increase Hcy-thiolactone synthesis by the plant
seedlings in the presence or absence of aminopterin (not shown).
Treatment of seedlings with aminopterin also increased the plant tissue
total Hcy level from 4.3 µM in the absence of aminopterin
to 245 µM in the presence of aminopterin. Hcy-thiolactone represented ~20% of total Hcy concentration in hypocotyls of yellow lupine seedlings grown in the presence of aminopterin. The growth of
6-day-old lupine seedlings was reduced in the presence of
aminopterin.
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Other antifolates, such as trimethoprim (0.1 mM) or sulfonamide (5 mM), did not reduce growth and did not affect Hcy-thiolactone or total Hcy levels in the plants. This suggests that trimethoprim, an inhibitor of bacterial dihydrofolate reductase enzymes (33), does not inhibit plant mitochondrial dihydrofolate reductase. The observation that Hcy or Hcy-thiolactone levels do not increase in the presence of sulfonamide, an inhibitor of de novo folate synthesis (32), suggests that endogenous methyltetrahydrofolate pools in lupine seedlings are not significantly depleted during growth. This suggestion is consistent with a study of one carbon fluxes in Arabidopsis thaliana, which indicated that cellular folate pools have a relatively long half-life in this plant (33).
Because of its mostly neutral character under physiological pH (18), Hcy-thiolactone is expected to diffuse out from lupine seedlings. Indeed, we have found that Hcy-thiolactone was excreted from seedlings grown in the presence of aminopterin. When yellow lupine seedlings were labeled with [35S]Hcy in the presence of aminopterin, the amount of [35S]Hcy-thiolactone excreted into medium in which seedlings were maintained represented 30% of the amount formed in hypocotyls (data not shown). In the absence of plant seedlings, no [35S]Hcy-thiolactone was formed in the aminopterin-containing medium.
MetRS Is Involved in the Synthesis of Hcy-thiolactone in
Plants--
To determine whether plant MetRS metabolizes Hcy to
Hcy-thiolactone, rice MetRS was expressed in E. coli BL21
harboring pET/MOsC, a plasmid bearing the rice MetRS gene under the
control of the lac promotor (25). The rate of
Hcy-thiolactone synthesis in the culture of E. coli
BL21/pET/MOs
C increased about 3-fold upon induction with
isopropyl-
-D-thiogalactopyranoside (Fig.
4), which indicates that rice MetRS
catalyzes the synthesis of Hcy-thiolactone. Supplementation of growth
medium with methionine resulted in inhibition of Hcy-thiolactone
synthesis in these cultures, as expected (Fig. 4).
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To determine whether MetRS is involved in Hcy-thiolactone synthesis in plants in vivo, yellow lupine seedlings were maintained on aminopterin in the presence of increasing concentrations of methionine. In the presence of 0.1 and 1 mM methionine, the plant tissue levels of Hcy-thiolactone dropped to 60% and 6%, respectively, of the levels observed in the absence of methionine (Table II). The inhibition by methionine is consistent with the involvement of MetRS in the synthesis of Hcy-thiolactone in lupine seedlings.
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Hcy Is Present in Yellow Lupine Proteins-- To determine whether Hcy is present in plant proteins, proteins from yellow lupine seedlings were extracted and depleted of free and disulfide forms of Hcy by treatments with DTT and precipitation with ethanol. Lupine proteins were then hydrolyzed with HCl in the presence of DTT. Under these conditions, Hcy, linked to protein by amide linkage (Hcy-N-protein), is converted to Hcy-thiolactone (10, 11, 26). As shown in Table I, the level of Hcy-N-protein in cotyledons of lupine seedlings increased after aminopterin treatment. Analysis of protein 35S-amino acids after labeling of seedlings with [35S]Met showed that [35S]Hcy-N-protein represented up to 5% of the [35S]Met-protein levels (Table I).
When seedlings were labeled with [35S]Hcy in the presence of aminopterin, the conversion of [35S]Hcy to [35S]Met-protein was inhibited 85%, compared with the conversion in the absence of aminopterin (Table III). The conversion of [35S]Hcy to [35S]Hcy-N-protein increased by 60% in the presence of aminopterin. [35S]Hcy-N-protein represented 8.3% and 87% of [35S]Met-protein in the absence and presence of aminopterin, respectively (Table III).
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Hcy-thiolacone Hydrolase Metabolizes Hcy-thiolactone in Yellow Lupine Plants-- To determine whether plants have the ability to metabolize Hcy-thiolactone, yellow lupine seedlings were maintained on 0.75 µM [35S]Hcy-thiolactone in water. The seedlings metabolized 60% and 100% [35S]Hcy-thiolactone after 7 and 24 h, respectively. Analysis of plant extracts showed that [35S]Met was a major metabolite derived from [35S]Hcy-thiolactone after 24 h (data not shown). In the absence of yellow lupine seedlings, Hcy-thiolactone was stable under the experimental conditions utilized (half-life > 3 days). Because Hcy-thiolactone is unlikely to be metabolized without ring opening, its fast metabolism suggests that Hcy-thiolactone-hydrolyzing enzyme is present in plants.
Indeed, when crude extracts from yellow lupine seeds were incubated with [35S]Hcy-thiolactone, it was hydrolyzed to [35S]Hcy at a rate of 0.21 µmol/mg/h. This level of Hcy-thiolactone-hydrolyzing activity is 3.7-fold higher than the level present in human serum (22). Lupine Hcy-thiolactonase activity, measured in extracts from cotyledons, did not change significantly after seed germination and growth up to 6 days (data not shown).
The Hcy-thiolactonase activity, precipitated from crude extracts of yellow lupine seed meal with 35% ammonium sulfate, was further purified by anion exchange chromatography on DEAE-Sephacel, gel exclusion chromatography on Superdex, and absorption chromatography on hydroxylapatite. At all steps of purification procedure, a single peak of Hcy-thiolactonase activity was observed, suggesting that a single enzyme was responsible for Hcy-thiolactone hydrolysis in yellow lupine. The specific activity of the purified Hcy-thiolactonase preparation, 536 µmol/mg/h, was 25,000-fold greater than that measured in crude extracts. The plant Hcy-thiolactonase preparation exhibited 7-fold higher specific activity than pure human Hcy-thiolactonase.
Examination of the substrate specificity showed that, in addition
to L-Hcy-thiolactone, the purified enzyme also hydrolyzed -aminoacyl esters and thioesters (Table
IV). For example, thioesters of
methionine, such as Met-S-CoA and Met-S-DTT, and
methionine methyl esters were hydrolyzed. Esters of other
-amino
acids, such as methyl esters of alanine, cysteine, phenylalanine,
tryptophan, and lysine, were also hydrolyzed.
D-Hcy-thiolactone and D-forms of
-aminoacyl
esters were hydrolyzed up to 20-fold less efficiently than the
L-forms. L-Homoserine-lactone was also a
substrate. However, N-acetyl-D,L-Hcy-thiolactone
was not hydrolyzed. Esters of
-amino acids, such as
-Ala
methyl ester, esters and thioesters of acetic acid, such as
O-acetyl-L-serine and acetyl-S-CoA,
and
-methyl ester of glutamic acid were not hydrolyzed. In contrast
to human Hcy-thiolactonase/paraoxonase, the plant enzyme did not
hydrolyze non-natural aryl esters, such as phenyl acetate and
p-nitrophenyl acetate, or the organophosphate paraoxon
(Table IV).
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The substrate specificity studies indicate that lupine
Hcy-thiolactone-hydrolyzing enzyme exhibits selectivity of an
-aminoacyl-(thio)ester hydrolase.
The plant Hcy-thiolactone-hydrolyzing enzyme eluted from a
Superdex gel filtration column as a 55-kDa protein. The enzyme exhibited a broad pH optimum, from pH 6 to pH 8, and did not require calcium or any other divalent cation for activity. The
Km value for L-Hcy-thiolactone was 45 mM. Taken together, these data indicate that the plant
enzyme is a novel Hcy-thiolactonase, fundamentally different from
human Hcy-thiolactonase/paraoxonase.
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DISCUSSION |
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This work demonstrates a novel aspect of Hcy metabolism in plants: synthesis and degradation of Hcy-thiolactone in the plant yellow lupine (Fig. 1). In the synthetic pathway, Hcy is converted to Hcy-thiolactone by the plant MetRS. In the degradation pathway, Hcy-thiolactone is hydrolyzed to Hcy by a unique plant Hcy-thiolactone hydrolase.
Hcy-thiolactone, a product of an error-editing reaction of MetRS in bacteria, yeast, and mammalian, including human, cells (2-17), has not been reported in plants before. As shown here, Hcy-thiolactone can be a significant component of sulfur amino acid pools in yellow lupine, particularly when the conversion of Hcy to methionine is limited by the antifolate drug aminopterin. Our results suggest that the synthesis of Hcy-thiolactone in plants is catalyzed by MetRS. The demonstration of Hcy-thiolactone synthesis also in plants supports a conclusion that Hcy editing during selection of amino acids for protein synthesis is most likely universal (15-17).
Hcy-thiolactone reacts easily with protein lysine residues under physiological conditions (20). This reaction is responsible for the presence of Hcy in endothelial cell proteins (11-15) and, most likely, in human blood proteins (26). Our data demonstrate that Hcy is also present in yellow lupine proteins. When methylation of Hcy to methionine synthase was inhibited by the antifolate drug aminopterin, Hcy-N-protein became a major metabolite of Hcy in yellow lupine seedlings. The presence of Hcy-N-protein in mammals (26) and plants suggests that Hcy-N-protein is likely to be a component of Hcy metabolism in multicellular organisms.
Incorporation of Hcy into protein mediated by Hcy-thiolactone is known
to result in protein damage (14-17, 20). Because of this, the ability
to detoxify Hcy-thiolactone is essential for biological integrity,
particularly in multicellular organisms. Indeed, specific
Hcy-thiolactone hydrolase/paraoxonase, tightly associated with high
density lipoprotein, exists in mammals, including humans (22, 23). Our
present work shows that yellow lupine plants possess a novel
Hcy-thiolactone-hydrolyzing enzyme. The plant Hcy-thiolactonase is
different from the human Hcy-thiolactone-hydrolyzing enzyme. For
example, the plant Hcy-thiolactonase does not require calcium for
activity, whereas the human Hcy-thiolactonase/paraoxonase does (22,
23). Although both enzymes hydrolyze Hcy-thiolactone, they differ in
their ability to hydrolyze other (thio)esters. For example, whereas the
plant enzyme hydrolyzes -aminoacyl (thio)esters, the human
enzyme does not (Table IV). On the other hand the plant enzyme does not
hydrolyze phenyl and p-nitrophenyl esters of acetic acid or
the organophosphate paraoxon. These artificial esters are very good
substrates of the human enzyme (Table IV).
In conclusion, our findings show that two novel pathways of Hcy
metabolism are utilized by the plant L. luteus: 1) metabolic conversion of Hcy to Hcy-thiolactone, a fundamental editing reaction in
protein synthesis, which appears to be conserved in all living organisms; and 2) hydrolysis of Hcy-thiolactone to Hcy, which thus far
has been documented in multicellular organisms such as mammals and plants.
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ACKNOWLEDGEMENTS |
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We thank S. Harvey Mudd (National Institutes
of Health, Bethesda, MD) for comments on plant sulfur metabolism, Marc
Mirande for a clone of rice MetRS, and Elbieta Starzy
ska
for help in purification of plant Hcy-thiolactonase.
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FOOTNOTES |
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* This research was supported by grants from the National Science Foundation, the National Research Council, the American Heart Association, and the Foundation of UMDNJ.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: Dept. of Microbiology and Molecular Genetics University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, International Center for Public Health, 225 Warren St., P. O. Box 1709, Newark, NJ 07101. Tel.: 973-972-4483 (ext. 28733); Fax: 973-972-8982; E-mail: jakubows@umdnj.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211819200
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ABBREVIATIONS |
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The abbreviations used are: Hcy, homocysteine; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; MetRS, methionyl-tRNA synthetase.
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