©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural and Functional Significance of Cysteine Residues of Glutathione-independent Prostaglandin D Synthase
IDENTIFICATION OF CYS AS AN ESSENTIAL THIOL (*)

(Received for publication, October 19, 1994)

Yoshihiro Urade (1) Toshiki Tanaka (2) Naomi Eguchi (1) Masami Kikuchi (2) Hiromi Kimura (2) Hiroyuki Toh (3) Osamu Hayaishi (1)(§)

From the  (1)Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565, Japan, the (2)Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan, and the (3)Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glutathione-independent prostaglandin D synthase in rat brain is composed of 189 amino acid residues and catalyzes the isomerization of prostaglandin H(2) to prostaglandin D(2), an endogenous sleep-promoting substance. This enzyme is the only enzyme among members of the lipocalin superfamily composed of various secretory lipophilic ligand-carrier proteins and is recently identified to be a beta-trace protein, a major constituent of human cerebrospinal fluid. We expressed the active enzyme in Escherichia coli and then systematically substituted all cysteine residues of the Delta1-29 enzyme at positions of 65, 89, and 186 with alanine or serine. The parent and mutant enzymes were purified to apparent homogeneity with a recovery of 30% by chromatography with Sephadex G-50 and S-Sepharose, by which all the enzymes showed identical elution profiles. The purified enzymes, irrespective of the mutation, showed almost the same circular dichroism spectral characteristics as displayed by a highly ordered beta-structure. The recombinant enzymes containing Cys showed the activity comparable with that of the enzyme purified from rat brain (3 µmol/min/mg of protein) in the presence, but not in the absence, of sulfhydryl compounds. However, all of the single, double, and triple mutants without Cys lost the enzyme activity. The purified Delta1-29 Ala enzyme was inactivated reversibly by conjugation with glutathione at Cys and irreversibly by the stoichiometric chemical modification with N-ethylmaleimide. These results indicate that Cys is an essential thiol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for nonoxidative rearrangement of 9,11-endoperoxide of prostaglandin H(2) to produce prostaglandin D(2) catalyzed by the enzyme.


INTRODUCTION

Prostaglandin (PG) (^1)D(2) is a major PG produced in the brain of rats and humans and functions as a neuromodulator of several central actions such as sleep-wake cycles, body temperature, luteinizing hormone release, and odor responses (for review see (1) ). PGD(2) induces natural sleep in rodents and monkeys after the intracerebroventricular administration (for review, see (2) ). Also in humans, accumulation of PGD(2) has been observed in the cerebrospinal fluid of patients with African sleeping sickness(3) . Therefore, PGD(2) is proposed to be an endogenous sleep-promoting substance in mammals.

Among several enzymes catalyzing the conversion of PGH(2) to PGD(2) (for review, see (4) ), GSH-independent PGD synthase, (5Z,13E)-(15S)-9alpha,11alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase (EC 5.3.99.2)(5) , is responsible for biosynthesis of PGD(2) in the brain. This enzyme is localized in the choroid plexus, leptomeninges, and oligodendrocytes of the central nervous system(6, 7) . It has been recently demonstrated that a major protein constituent of human cerebrospinal fluid, classically termed beta-trace, is identical to PGD synthase(8, 9) . Concurrently, sleep of unrestrained rats has been found to be inhibited by the intracerebroventricular administration of selenocompounds(10) , which inhibit the rat brain PGD synthase activity without affecting the activities of three other enzymes in the arachidonate cascade, GSH-requiring PGD synthase, PGF synthase, and 11-keto PGD(2) reductase(11) . Furthermore, when PGD(2) was infused into the subarachnoid space near the ventral surface of the rostral basal forebrain, the sleep-promoting effect of PGD(2) in rats was found to be more remarkable than the intracerebroventricular administration or the infusion into the brain parenchyma(12) . Thus, the enzyme is considered to play a key role in the regulation of sleep-wake activities in mammals(13) , probably via the cerebrospinal fluid.

We have already reported the cloning and sequence analyses of the rat and human cDNAs (14, 15) and the rat gene (16) for the enzyme. White et al. (17) have isolated the human gene for the enzyme and mapped the gene to chromosome 9, bands q34.2-34.3. A homology search in data bases of protein primary structure revealed that the enzyme is a member of the lipocalin superfamily(15, 18, 19) , a group of proteins comprising a variety of secretory proteins that bind and transport small lipophilic molecules(20) . PGD synthase is the only known exception, being an enzyme rather than a lipophilic ligand-carrier protein. Based on the observed sequence homology, the enzyme was deduced to form an eight- or nine-stranded anti-parallel beta-barrel structure with a hydrophobic pocket in which Cys is located(15) . Among members of the lipocalin family, the cysteine residue is conserved only in rat and human brain PGD synthases and in a major secretory protein from amphibian choroid plexus(21) , the latter of which has the highest homology to the enzyme. Therefore, Cys has been assigned to be a putative active site of this enzyme(15, 18) .

In this study, we investigated the structural and functional significance of cysteine residues of GSH-independent PGD synthase by site-specific mutagenesis and chemical modification. Our results demonstrate that Cys is an essential thiol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for the catalysis effected by this enzyme. The reaction mechanism of this enzyme is also proposed.


MATERIALS AND METHODS

Expression of Recombinant Rat Brain PGD Synthase in Escherichia coli

An EcoRI insert (0.8 kilobase pairs) from a gt 11 clone, which contains a full-length cDNA of rat brain PGD synthase composed of 189 amino acid residues (M61900)(15) , was ligated into the multiple cloning sites of the expression vector pUC119. The vector construct was then used to infect E. coli JM109. Transformants carrying the cDNA insert in the correct orientation were selected by a restriction enzyme analysis of the plasmid DNA with both BamHI and PstI. The plasmid was cleaved with BamHI/SphI and then partially digested with exonuclease III at 25 °C for various periods of time (4-20 min). The digested plasmid was incubated with Mung bean nuclease followed by T4 polymerase, self-ligated with T4 DNA ligase (Kilo-Sequence deletion kit, Takara Shuzo, Kyoto, Japan), and then employed for infection of E. coli. The deletion mutants with a correct coding frame were immunoscreened with polyclonal antibodies against PGD synthase purified from rat brain (3) to obtain a series of the recombinant enzyme of a total of 40 different mutants lacking various sizes of the N-terminal sequence with about 5-amino acid intervals.

Site-specific Mutagenesis

The C-terminal truncated mutants were produced by introducing a stop codon (TAA) into the desired positions of the cDNA and removing the downstream coding sequence. The site-specific mutagenesis was achieved by nucleotide replacement of the codon for Cys to that of Ala (GCC) or Ser (TCT). The mutated cDNAs were constructed by the polymerase chain reaction (22) with primers of 25-30 base pairs long that contained the replaced codon in a center portion. The nucleotide sequence was confirmed by DNA sequencing by the dideoxynucleic acid sequencing method(23) .

Purification of Recombinant Rat Brain PGD Synthase

E. coli transfected with the reconstructed plasmid were cultured at 37 °C overnight in 500 ml of LB medium in the presence of 2.5 mM isopropyl-1-thio-beta-D-galactopyranoside. The cells were harvested, sonicated at 4 °C in a 10-fold volume of 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and 10 mM dithiothreitol (DTT), and centrifuged at 20,000 times g for 30 min at 4 °C. The protein in the supernatant was precipitated in the presence of 80% saturated ammonium sulfate, redissolved in 15 ml of the buffer solution containing 10 mM DTT, and applied to a Sephadex G-50 column (bed volume, 500 ml) equilibrated beforehand with 20 mM Tris-HCl (pH 7.5) containing 50 mM NaCl, 1 mM EDTA, and 1 mM DTT. The fractions containing the enzyme were collected and adjusted to pH 4.5 by the addition of acetic acid. After centrifugation to remove denatured proteins, the supernatant was loaded onto an S-Sepharose column (bed volume, 30 ml) in 10 mM sodium citrate (pH 4.5) containing 1 mM EDTA and 1 mM DTT. The column was washed and then eluted with an NaCl gradient (0-0.8 M). The oxidized isoforms of the enzyme were purified in the absence of DTT by the same procedures as described above. The elution profile of the enzyme was monitored by measuring the enzyme activity and immunoblotting analysis as described below.

Enzyme Assays

The PGD synthase activity was measured by incubation at 25 °C for 1 min with [1-^14C]PGH(2) (final concentration, 40 µM) in 50 µl of 0.1 M Tris-HCl (pH 8.0) in the presence of 1 mM DTT unless otherwise stated(3) . [1-^14C]PGH(2) was prepared from [1-^14C]arachidonic acid (2.20 GBq/mmol; DuPont NEN) as described previously(3) . Protein concentration was determined by the method of Lowry et al. (24) as modified by Bensadoun and Weinstein(25) .

Immunoblotting

For Western blot analyses, E. coli lysates or the purified recombinant enzyme were subjected to polyacrylamide gel electrophoresis (PAGE) after solubilization with 1% SDS in the presence or absence of 1% beta-mercaptoethanol. The proteins were electrophoretically transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA). The membranes were immunostained with polyclonal rabbit anti-rat brain PGD synthase antibodies (3) and alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Organon Teknika, Durham, NC).

Circular Dichroism

CD spectra were recorded on a J-720 spectropolarimeter (Japan Spectroscope, Tokyo, Japan) with the sample in a 1-mm path length cuvette at 10 °C. The spectrometer was calibrated with ammonium d-(+)-10-camphor sulfonate at 290.5 nm and with d-(-)-pantoyllactone at 219 nm. The mean residue ellipticity, Q, which has the units of deg cm^2 dmol, was calculated by using a molar absorption coefficient of the enzyme, , of 25,131 M cm. The content of the secondary structure components was calculated by the method of Provencher and Glöckner(26) .

Sulfhydryl Modification

A given amount of the purified enzyme was incubated at 25 °C for 5 min with various amounts of N-ethylmaleimide (NEM) in 25 mM Tris-HCl (pH 7.0) containing 1 mM EDTA. After incubation, the reaction mixture was divided into two halves. One half was used for determining free sulfhydryl content of the enzyme after incubation at 25 °C for 5 min with 400 µM 5, 5`-dithio-bis-(2-nitrobenzoic acid) by the method of Ellman(27) . The other half was used for determining the residual enzyme activity after addition of an excess amount of DTT to quench the sulfhydryl modification.

For reversible modification of the cysteine residue, the enzyme was incubated with a mixture of each 1 mM of GSH and the oxidized form of GSH in 0.1 M Tris-HCl (pH 9.0) and 0.1 M KCl containing 1 mM EDTA. After the incubation at 4 °C overnight, the cysteine residue was autooxidized to form a GS-adduct. The modification was confirmed by the decrease in the free sulfhydryl content of the enzyme, and the increase in M(r) of the enzyme was determined by time of flight mass spectrometry after matrix-assisted laser-desorption/ionization (Maldi-Tof mass monitor LDI-1700, Jeol, Tokyo, Japan). The modification was removed by incubation at 4 °C overnight with excess DTT.

Multiple Alignment

A multiple sequence alignment was obtained by the randomized iterative algorithm (28) among rat and human PGD synthases(14, 15) , the secretory protein of cane toad choroid plexus(21) , bovine beta-lactoglobulin(29) , and human plasma retinol-binding protein(30) . The iteration was initiated from an alignment constructed by the tree-based algorithm (31) and repeated 500 times. McLachlan's score matrix (32) was used for the alignment. The aligned positions of six motifs highly conserved among the lipocalin family (20) were fixed during the calculation.


RESULTS AND DISCUSSION

Expression of Recombinant Rat Brain PGD Synthase after Sequential Truncation from N and C Termini

cDNA for rat brain PGD synthase was unidirectionally deleted from 5` terminus and incorporated into a PUC 119 expression vector. After infection of E. coli with the reconstructed plasmids after N-terminal deletion up to 29 amino acid residues, the enzyme of various sizes was expressed in the soluble fraction. Further deletions resulted in integration of the expressed protein into an inclusion body or its degradation within the cell. Typical results are shown in Fig. 1A. The PGD synthase activity was detected in the soluble fraction of Delta1-19, Delta1-23, and Delta1-29 mutants (0.58, 0.65, and 0.60 µmol/min/mg of protein, respectively) but not of the shorter mutants such as Delta1-34 and Delta1-47 mutants (<0.01 µmol/min/mg of protein). In the precipitate fraction of all samples, no enzyme activity was detected. When we removed a sequence of Thr-Met-Ile-Thr-Pro-Ser-Leu coded in a linker portion of the plasmid from the Delta1-29 enzyme, the amount of the enzyme produced in E. coli was significantly decreased (data not shown). Therefore, the Delta1-29 enzyme containing the 7 additional residues was used for the further mutagenesis studies.


Figure 1: SDS-PAGE of E. coli lysates containing various sizes of recombinant rat brain PGD synthase after sequential truncation. The amino acid residues of PGD synthase were unidirectionally deleted from the N terminus (A) and the C terminus (B). E. coli transfected with the mutated plasmid were sonicated at 4 °C in a 10-fold volume of 20 mM Tris-HCl (pH 7.5) and 1 mM EDTA and centrifuged at 20,000 times g for 30 min at 4 °C. The precipitated (P) (each leftlane) and soluble (S) (each rightlane) fractions were subjected to SDS-PAGE under reducing conditions. Proteins in the gel were stained with Coomassie Blue. M(r) values in thousands of marker proteins are indicated on the left.



We then constructed C-terminal truncated mutants by introducing the stop codon into the positions of the amino acid residues of 154, 171, and 183 of the Delta1-29 enzyme. The Delta(1-29, 183-189) enzyme was efficiently, but partly, expressed into the soluble fraction and associated with the enzyme activity (0.42 µmol/min/mg of protein). However, the shorter mutants, the Delta(1-29, 171-189) and Delta(1-29, 154-189) enzymes, were completely recovered in the insoluble fraction (Fig. 1B) and had no enzyme activity (<0.01 µmol/min/mg of protein). Thus, the Delta(1-29, 183-189) mutant was an active enzyme of the smallest size produced by terminal truncation, being composed of 153 amino acid residues from the cDNA plus 7 residues from the plasmid with a calculated M(r) of 18,136. The core region from Val to Gln, essential for stable expression of the recombinant enzyme with the catalytic activity, ranges from upstream of the first beta-strand (A-strand) to downstream of the last beta-strand (I-strand) assigned in the multiple alignment with other members of the lipocalin family (Fig. 2).


Figure 2: Multiple alignment of amino acid sequences of rat and human brain PGD synthases, a major secretory protein from toad choroid plexus, bovine beta-lactoglobulin, and human retinol-binding protein. Cleavage sites of the signal sequences of these proteins (arrowheads) and N-glycosylation sites of PGD synthase (asterisks) are shown on the top of each sequence. Vertical boxes represent the cysteine residue conserved among PGD synthases and the toad protein (open box), whose residue was identified as an active thiol of rat brain PGD synthase, and 2 cysteine residues constructing an intramolecular disulfide bond highly conserved among members of the lipocalin superfamily (patched boxes). The positions of secondary structure elements of beta-lactoglobulin (33) and retinol-binding protein (34) identified by x-ray crystallography are shown at the bottom of each sequence: 3 helix, a hatched box; alpha-helix, open boxes; and beta-strand, closed boxes. In both proteins, beta-strands B, C, and D, and F, G, and H construct two anti-parallel beta-sheets forming a beta-barrel structure(33, 34) .



Recently the N-terminal amino acid sequence of rat brain PGD synthase was determined to be Gly-His-Asp-Thr-Val-Gln-Pro after digestion with pyroglutamyl aminopeptidase, indicating that Gln is the N terminus of the mature enzyme. (^2)PGD synthase in human cerebrospinal fluid, beta-trace, is also processed posttranslationally, with the first N-terminal 22 amino acid residues being cleaved(7, 8, 15) . Therefore, the N-terminal sequence is considered to function as a signal peptide for its sorting into the membrane (5) and the secretion into the cerebrospinal fluid. These results agree with the finding that the N-terminal sequence upstream of Val was not essential for the activity of the enzyme. PGD synthases purified from rat brain (14) and human cerebrospinal fluid (7) are N-glycosylated at two positions, Asn and Asn. However, since the recombinant enzyme displayed the enzyme activity, the glycosyl residues are also not considered to be essential for the enzyme activity.

Expression of Recombinant Rat Brain PGD Synthase after Systematic Cys Substitution

We then systematically replaced all cysteine residues at three positions of 65, 89, and 186 of the Delta1-29 enzyme with alanine or serine to examine the structural and catalytic significance of these residues. In the multiple alignment (Fig. 2), Cys is located in or at the edge of the B-strand, Cys in the D-strand, and Cys in a tail sequence downstream of the I-strand. Two cysteine residues corresponding to Cys and Cys of the enzyme are highly conserved among members of the lipocalin superfamily and construct an intramolecular disulfide linkage. As judged by SDS-PAGE of the lysates under reducing conditions, all mutant enzymes were efficiently expressed in E. coli to be a major protein and a single immunoreactive protein with anti-rat brain PGD synthase antibodies at a position of M(r) of 20,000. Alternatively, after SDS-PAGE under nonreducing conditions, various isoforms of the enzyme were observed in the lysates. Typical results for replacement by alanine are shown in Fig. 3.


Figure 3: Western blot analysis after SDS-PAGE of E. coli lysates expressing recombinant rat brain PGD synthase after Cys-Ala substitution. Samples are the lysates of transformed E. coli expressing the recombinant Delta1-29 PGD synthase and the mutant enzymes after Cys-Ala substitution at three positions. The samples were denatured in the presence (+) or the absence(-) of beta-mercaptoethanol (SH). The gel was stained with silver (each rightlane), and the transfer blot was immunostained with antibodies against rat brain PGD synthase (each leftlane). Symbols represent the recombinant enzymes of a monomer with (open arrowheads) and without (closed arrowheads) intramolecular disulfide linkage, a monomer after endogenous sulfhydryl modification (asterisks), dimers (small arrows), and oligomers (dotted lines). Positions of the M(r) marker proteins are shown by horizontal bars at the left.



Major components of nonreduced isoforms of the parent, Ala, Ser, Ala, and Ser enzymes migrated in the gel faster than the reduced forms. These isoforms possessed intramolecular disulfide bonds: a Cys-Cys linkage in the Ala and Ser enzymes, a Cys-Cys linkage in the Ala and Ser enzymes, and both linkages in the parent enzyme. These results suggest that Cys is accessible to both Cys and Cys probably due to the location of Cys in a flexible C-terminal sequence of the enzyme. In Western blot, the enzymes with the Cys-Cys bond were less immunoreactive than those with the Cys-Cys bond, suggesting that the antigenic epitopes of the enzyme were masked by the formation of the Cys-Cys disulfide bond. Alternatively, the intramolecular disulfide linkage was not found between Cys and Cys of both Ala and Ser mutants, indicating that these 2 residues are kept at a distance from one another in the enzyme molecule. This finding is also consistent with the model structure of the enzyme (14) in which three beta-strands, the Cys-containing B-strand, the Cys-containing D-strand, and the C-strand, construct a beta-sheet structure, and the former two beta-strands are separated by the C-strand in the same beta-sheet structure.

For all mutants without Cys, the major component existed as an isoform without disulfide bridge and migrated to a position identical to that seen under reducing conditions. The Cys-possessing enzymes, but not other mutants without Cys, showed an additional major band at a position of slightly higher M(r). Since the additional band disappeared after incubation with excess DTT, it was considered to be due to sulfhydryl modification of Cys that endogenously occurred during expression of the enzyme in E. coli. The modification was not found at other 2 cysteine residues, Cys and Cys. Thus, Cys is an active thiol of the enzyme in terms of the endogenous modification.

Intermolecular disulfide formation was observed in all enzymes except for the Ala enzyme without cysteine residues. The parent and all single mutants formed dimers, trimers, and oligomers via intermolecular disulfide bonds among the available cysteine residues. The double mutants formed only dimers via an intermolecular disulfide bond at a unique cysteine residue of each mutant. These dimers and oligomers were completely dissociated into the corresponding monomers after incubation with excess DTT. Among the double mutants, the ratio of a dimer to a monomer was higher in the Ala and Ala enzymes than in the Ala enzyme, suggesting that the disulfide bond is formed more preferentially at Cys and Cys than at Cys. Therefore, Cys and Cys may be exposed on the surface of the enzyme molecule to a greater extent than Cys. It is in agreement with the predicted tertiary structure of the enzyme(14) , in which Cys exists in the hydrophobic pocket of the enzyme and an intramolecular disulfide bridge between Cys and Cys occurs at the outside of the barrel structure.

Purification and Characterization of Recombinant Rat Brain PGD Synthase

To determine the PGD synthase activity of the parent and mutant enzymes, they were purified to apparent homogeneity by Sephadex G-50 and S-Sepharose chromatographies under reducing conditions in the presence of 1 mM DTT. Typical results of purification of the Delta1-29 Ala enzyme are summarized in Fig. 4A and Table 1. The elution profiles and recoveries from these two chromatographies were almost the same among all recombinant enzymes; the enzyme was eluted with a peak at a position of M(r) of 20,000 from Sephadex G-50 column and at NaCl concentrations of 0.4 M from S-Sepharose column. These results indicate that the tertiary structure of the enzyme in the buffer solution remained unchanged significantly by substitution of the cysteine residues.


Figure 4: Purification of the reduced form of the Delta1-29 Ala mutant (upper panels) and the nonreduced forms of the Delta1-29 Ala mutant (lower panels). Elution profiles of protein (solid lines) and the PGD synthase activity (open circles) were monitored during column chromatographies with Sephadex G-50 (A) and S-Sepharose (B). The PGD synthase activity of the nonreduced isoforms was also determined after incubation of the eluate at 4 °C overnight with excess DTT in 0.1 M Tris-HCl (pH 8.0) (closed circles in lowerpanels). The position of the void volume (V in A) and the gradient of NaCl concentrations (dotted line in B) are also indicated. Horizontal bars represent the collected fractions.





The oxidized isoforms of the Ala and Ser enzymes with the intramolecular Cys-Cys linkage were also purified to apparent homogeneity in the absence of DTT, giving almost the same elution profiles and recoveries as those of the reduced isoforms in the presence of 1 mM DTT. The findings suggest that the tertiary structure of the enzyme was unchanged by formation of the intramolecular disulfide linkage.

Alternatively, in the absence of DTT, other mutants showed heterogeneous peaks of various isoforms in the two chromatograms. The elution profiles of the Ala enzyme purified in the absence of DTT are shown in Fig. 4B. The dimeric and oligomeric isoforms with the intermolecular disulfide bond were eluted at positions of higher M(r) from Sephadex G-50 column. The monomeric isoforms with the endogenously modified Cys and the intramolecular Cys-Cys linkage were eluted from S-Sepharose column with peaks at NaCl concentrations of about 0.2 and 0.6 M, respectively. These results revealed that those isoforms were distict from others in terms of the net charge at pH 4.5; the former isoform was charged less positively and the latter more positively than other isoforms. From the difference of M(r) between the modified and unmodified enzymes, M(r) of the endogenous modifier was calculated to be about 800, although the chemical structure remains unidentified. Typical results of SDS-PAGE of the monomeric isoforms purified under the nonreducing conditions are shown in Fig. 5.


Figure 5: SDS-PAGE of the monomeric isoforms of the Delta1-29 Ala, Ala, and Ala enzymes purified under nonreducing conditions. Each sample was subjected to SDS-PAGE under nonreducing conditions before (A) and after (B) incubation with excess DTT and then stained with Coomassie Blue. Samples are the Delta1-29 Ala enzyme without endogenous sulfhydryl modification at Cys (lane 1), the Delta1-29 Ala enzyme with an intramolecular Cys-Cys disulfide bond (lane 2), the Delta1-29 Ala enzyme with an intramolecular Cys-Cys disulfide bond (lane 3), the Delta1-29 Ala enzyme without the disulfide bond and the endogenous modification at Cys (lane 4), and the Delta1-29 Ala enzyme with the endogenous modified Cys (lane 5). The marker proteins are shown at the left.



Fig. 6A shows the CD spectrum of the purified Delta1-29 Ala enzyme, which revealed characteristics of a beta-sheet structure (35) with a broad minimum peak at around 214 nm, and was remarkably similar to that of beta-lactoglobulin(36) . The content of secondary structural components of the enzyme was calculated to be 17% alpha-helix, 47% beta-strand, and 36% unordered structure. The parent and all of the other mutant enzymes showed the same CD spectra as that of Delta1-29 Ala enzyme, in which the oxidized isoforms of the Ala and Ala enzymes with the intramolecular Cys-Cys and Cys-Cys linkage, respectively, were also included. Furthermore, the CD spectrum remained unchanged when the disulfide bond of these isoforms was cleaved by incubation with excess DTT. These results also indicate that the tertiary structure of the enzyme is unchanged by the formation of the disulfide linkage.


Figure 6: Circular dichroism spectra of the purified recombinant rat brain PGD synthase. The samples are the Delta1-29 Ala mutant purified in the presence of 1 mM DTT (A), and the Delta1-29 Ala mutant with an endogenously modified Cys purified in the absence of DTT (B). The spectrum of the modified enzyme was also measured after removing the modification by the incubation with excess DTT (dottedline in B). The spectrum was obtained as the mean of 10 recordings with the protein solution at a concentration of 15 µM in 5 mM potassium phosphate (pH 7.5).



However, as shown in Fig. 6B, the CD spectrum of the isoform with the endogenously modified Cys was clearly different from that of other isoforms, suggesting that the modifier has the minimum value at about 220 nm. The CD spectrum of the modified enzyme returned to that of the unmodified enzyme after incubation with excess DTT, which removed the modification to produce the free sulfhydryl group.

Identification of Cys as an Essential Thiol of Rat Brain PGD Synthase

The PGD synthase activity of the reduced form of the purified Delta1-29 enzyme and its single and double mutants containing Cys was comparable with that of the enzyme purified from rat brain (3 µmol/min/mg of protein, Table 2). All of these active enzymes required sulfhydryl compounds, such as DTT, beta-mercaptoethanol, glutathione, or cysteine, for catalysis. In the absence of sulfhydryl compounds, the enzymes were completely inactive (<0.01 µmol/min/mg of protein), similar to the enzyme purified from rat brain(3) . Alternatively, even in the presence of sulfhydryl compounds, the PGD synthase activity was not detected in any of the single, double, and triple mutants lacking Cys (<0.01 µmol/min/mg of protein, Table 2). The PGD synthase activity was neither detected in the Delta1-29 Ala and Delta1-29 Ala enzymes containing the endogenously modified Cys residue, the Delta1-29 Ala enzyme with an intramolecular Cys-Cys bond, nor the Delta1-29 Ala enzyme conjugated with GSH to form the GS-adduct at Cys (<0.01 µmol/min/mg of protein). All of these inactive enzymes became active (3 µmol/min/mg of protein) after incubation with excess DTT, which reproduced a free sulfhydryl group of Cys.



When the purified Delta1-29 Ala enzyme was incubated with various amounts of NEM, the enzyme activity and the content of free sulfhydryl group of the enzyme decreased dose-dependently in a parallel fashion and disappeared almost completely after incubation with excess amounts of NEM (Fig. 7). These results indicate that the inactivation of this enzyme was due to stoichiometric chemical modification of Cys by NEM and that Cys is an essential thiol of the enzyme.


Figure 7: Effects of chemical modification with NEM on the enzyme activity and the free sulfhydryl content of the purified Delta1-29 Ala PGD synthase. The enzyme (7 nmol) was incubated at 25 °C for 5 min in the presence or absence of various amounts of NEM in 240 µl of 25 mM Tris-HCl (pH 7.0) and 1 mM EDTA. After incubation, the residual enzyme activity (open circles) and the free sulfhydryl content of the enzyme (closed circles) were determined as described under ``Materials and Methods.'' The free sulfhydryl content of the enzyme before incubation was determined to be 0.8 mol/mol enzyme.



The Cys residue is conserved among rat and human brain PGD synthases and a major secretory protein from cane toad choroid plexus (21) but is not present in any of the other members of the lipocalin superfamily(15, 18, 19) . The toad secretory protein has the highest homology with PGD synthase among lipocalins identified to date; the identity and similarity are 41.4 and 66.1% to the rat enzyme and 39.8 and 64.5% to the human enzyme, respectively (Fig. 2). The toad protein and PGD synthase also share several common features for the tissue distribution; both the protein and the enzyme are selectively expressed in the central nervous system, actively produced in the choroid plexus, and secreted into the cerebrospinal fluid(6, 7, 8, 21) . The toad protein may, therefore, be an amphibian counterpart of the mammalian enzyme and may also be associated with the PGD synthase activity, acting as the enzyme in the central nervous system of amphibians.

In conclusion, our results have shown that Cys is an essential thiol for the activity of PGD synthase and that both intrinsic and extrinsic sulfhydryl groups are necessary for catalyzing the conversion of PGH(2) to PGD(2). The extrinsic sulfhydryl group was required for the reaction but not consumed stoichiometrically during the reaction. After the conversion of PGH(2) to PGD(2), configuration of chiral carbon at C-9 is retained. Therefore, as shown in Fig. 8, PGD(2) formation is considered to be initiated by the transient formation of an S-O linkage between the active intrinsic thiol of Cys and the oxygen at C-11 of PGH(2). The extrinsic sulfhydryl group then withdraws the hydrogen at C-11 of the intermediate to produce PGD(2). PGD synthase is known to be rapidly inactivated during reaction(3) . The suicidal property of this enzyme might be due to nucleophilic attack of the extrinsic sulfhydryl group toward the carbon at C-11 instead of the hydrogen, as shown by dottedarrows in Fig. 8. In consequence, PGD synthase may be inactivated by oxidation of the intrinsic thiol of Cys.


Figure 8: Proposed mechanism of a nonoxidative rearrangement of PGH(2)to PGD(2) catalyzed by PGD synthase in the presence of exogenous sulfhydryl compounds. The cyclopentane ring structure of PGs is shown. Dotted arrows show the putative inactivation process of PGD synthase during the reaction. RSH represents exogenous sulfhydryl compounds.



The reaction mechanism may also be applicable to PGE synthase because this enzyme shows several enzymatic characteristics identical to PGD synthase(37, 38, 39) . For example, both of the enzymes are sensitive against sulfhydryl modifiers, require the extrinsic sulfhydryl compound as an essential cofactor, and are rapidly inactivated during the reaction. The extrinsic sulfhydryl compound is not oxidized stoichiometrically during the reactions catalyzed by these two enzymes. Thus, the reaction mechanism of PGE synthase may be the same as that of PGD synthase and different from the previously speculated mechanism analogous to the glyoxylase I reaction(40) . PGE, PGI, and thromboxane A synthases in the arachidonic acid cascade also catalyze the nonoxidative rearrangement of 9,11-endoperoxide of PGH(2). Since the reaction mechanisms of these enzymes are still a matter of speculation, the proposed mechanism in this study for PGD synthase is useful to predict their reaction mechanisms.


FOOTNOTES

*
This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan (to N. E. and O. H.), the Uehara Memorial Foundation (to Y. U.), the Japan Foundation for Applied Enzymology (to Y. U.), and the Mitubishi Foundation (to Y. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed.

(^1)
The abbreviations used are: PG, prostaglandin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; NEM, N-ethylmaleimide.

(^2)
Y. Urade, N. Eguchi, and T. Tanaka, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. A. Nagata, Tokyo University, for guidance during the early stage of this study and Dr. S. Sri Kantha for critical reading of the manuscript. We also thank H. Kondoh, E. Kitakuni and I. Umemura for technical assistance and K. Sugimoto for secretarial assistance.

Note Added in Proof-Ullrich and Brugger (41) have recently proposed the models for the reaction mechanism of PGI and thromboxane A synthases.


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