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J. Biol. Chem., Vol. 280, Issue 15, 14884-14891, April 15, 2005
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From the
Departments of Molecular Biology & Pharmacology and ¶Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 and the Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843
Received for publication, December 22, 2004 , and in revised form, January 20, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I0.5 = 30 µM. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In some organisms, such as Escherichia coli (7), PGDH is strongly inhibited by L-serine (I0.5 =
2–4 µM), the end product of the pathway. E. coli PGDH is the most studied and is classified as a V-type enzyme (8) because L-serine regulates catalysis by altering the velocity of the reaction rather than the affinity of the substrate. In Bacillus subtilis, inhibition of PGDH by L-serine is less sensitive (I0.5 0.6 mM) but appears to lose its sensitivity to L-serine under oxidizing conditions (9). PGDH from Corynebacterium glutamicum has also been reported to be inhibited by L-serine only at very high concentrations (I0.5 10 mM) but has not been studied in homogeneous form (10). In addition, L-serine inhibition of both the B. subtilis and C. glutamicum PGDH require extensive preincubation of the enzyme with the inhibitor before appreciable inhibition can be measured. In the pea (Pisum sativum), the sensitivity to L-serine has been reported to be cold labile (3). In crude extracts of wheat germ (4), PGDH activity appears to be inhibited by high concentrations of L-serine, but this sensitivity is reported to be lost upon incubation, and the purified wheat germ PGDH is not inhibited by L-serine. L-Serine has been reported to have no effect on the activity of PGDH from rat liver (11) and chicken liver (12). The effect of L-serine on the activity of PGDH from other species has not been reported.
PGDH exists in at least three different basic structural motifs that do not appear to be strictly specific for organism type (11). The PGDH of some bacteria and some lower eukaryotes, such as yeast, Leishmania, and Neurospora, are structurally similar to the E. coli enzyme (Figs. 1 and 2). In addition to substrate and nucleotide-binding domains, they possess a homologous C-terminal domain that, in E. coli, is involved in effector binding (L-serine) and regulation of activity. This third domain has been called the "ACT domain" and consists of a 
structural motif that has been found in other proteins as well (13, 14). Other bacteria and higher order eukaryotes, including mammals, possess a large polypeptide insertion in their C-terminal segment immediately following the substrate-binding domain. Furthermore, the homology to the ACT domain following this insertion is minimal. A third motif, which lacks the C-terminal regulatory domain altogether, is also found in some bacteria, including some, such as Mycobacterium tuberculosis, that also produce PGDH with the extended C-terminal motif. Recently, a variation of this third motif has been recognized in the parasite Entamoeba histolytica (15) that appears to utilize a lysyl residue rather than a histidyl residue at the active site for proton transfer.
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PGDH from M. tuberculosis contains an extended C-terminal domain like that found in mammalian PGDH, but we have observed that it is readily inhibited by L-serine. In fact, it is the only other purified PGDH that has been shown to be inhibited by L-serine. Although it has not yet been characterized, the human enzyme is expected to have properties similar to the rat PGDH, which is insensitive to L-serine, because they are 94% identical.
Tuberculosis is a deadly disease that is prevalent throughout much of the world and that is showing significant development of multidrug resistance. If the human enzyme is, in fact, insensitive to L-serine, this may provide a possible avenue toward the development of new drugs to treat tuberculosis. This study reports the expression and characterization of L-serine-sensitive PGDH from M. tuberculosis and demonstrates that it possesses a unique mode of regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning, Expression, and Purification of PGDH from M. tuberculosis—The SerA1 gene, Rv2996c, from the M. tuberculosis H37Rv genome, was identified from the Tuberculist data base. The SerA1 gene was amplified using the H37Rv genomic DNA as a template. The PCR product was cloned into the pET 30b vector by insertion into the NdeI and HindIII sites. The NdeI site contained the ATG start codon, and the Hind III site was preceded immediately by a stop codon following the codon for the C-terminal residue of the enzyme.
The culture is grown in LB medium with kanamycin (50 µg/ml) at 37 °C until the A600 nm reaches 0.6–0.8. PGDH expression is induced with 1 mM isopropyl -D-thiogalactopyranoside and allowed to grow at 37 °C for 6–7 h. The cells are harvested by centrifugation at 2,000 x g for 30 min and resuspended in 20 mM potassium phosphate, pH 7.5, 5 mM KCl. After the addition of lysozyme (0.16 mg/ml), the cells are disrupted by sonication for 2–3 min. Cell debris is removed by centrifugation at 27,000 x g for 1 h, and the supernatant is dialyzed against 20 mM potassium phosphate, pH 7.5, 5 mM KCl in preparation for ion exchange chromatography. A 2.6 x 8-cm column of Q-Sepharose is equilibrated in 20 mM potassium phosphate, pH 7.5, 5 mM KCl. After the sample is applied to the column, the column is washed with starting buffer until the A280 reaches approximately 0. The protein is eluted with a 1200-ml linear gradient of 25 to 1000 mM KCl in 20 mM potassium phosphate, pH 7.5, and the fractions are collected. M. tuberculosis PGDH is located by assaying for enzymatic activity, and the appropriate fractions are pooled. The pooled fractions are dialyzed against 100 mM potassium phosphate, pH 7.5, 5 mM KCl and rechromatographed on an identical Q-Sepharose column with a linear gradient from 25 to 1000 mM KCl.
Sequence Homology—All of the sequences were obtained from the National Center for Biotechnology Information ENTREZ data base (www.ncbi.nlm.nih.gov/). Sequence alignments were initially performed using the SIM program from the ExPASY Molecular Biology server at us.expasy.org/tools/.
Serine Inhibition and Binding—Serine inhibition plots were fit to the Hill equation as previously described (17). Binding of L-serine to M. tuberculosis PGDH was performed as described previously (18) using tritiated L-serine. Stepwise intrinsic dissociation constants for L-serine were determined by fitting to the Adair equation for four binding sites as previously described. Hill plots were utilized to determine the dissociation constant for the binding of the first serine (19). This was subsequently used to constrain K1' in the Adair equation. All of the data were fit with Kaleidograph version 3.6 from Synergy Software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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55,000 that is consistent with the molecular weight predicted from its sequence (Fig. 3).
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-ketoglutarate as a substrate (20). Neither substrate produces inhibition of E. coli PGDH activity at high concentrations. In contrast, M. tuberculosis PGDH is specific for hydroxypyruvic acid phosphate as a substrate as was also demonstrated for rat liver PGDH. Neither enzyme displays any activity with -ketoglutarate. In addition, M. tuberculosis PGDH exhibits substrate inhibition (Fig. 4) similar to what has been reported for rat liver PGDH (11). Utilizing the initial part of the curve in a v versus S plot and fitting it to the Michaelis-Menten equation, the Km for hydroxypyruvic acid phosphate is 85 µM, and the kcat/Km is 5.6 x 106 M–1 s–1. The E. coli enzyme has a Km of 40 µM and a kcat/Km of 7 x 105 M–1 s–1 for hydoxypyruvic acid phosphate. For -ketoglutarate the E. coli enzyme has a Km of 0.5 mM and a kcat/Km of 3 x 10 4 M–1 s–1. The pH at which M. tuberculosis PGDH displays optimal activity is 6.5, whereas that for E. coli PGDH is 8.5. Although M. tuberculosis PGDH utilizes NADH to catalyze the same reaction as the E. coli and rat liver enzymes, it is not retained on a column of 5'AMP-Sepharose as they are. These characteristics are summarized in Table I.
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75–100 mM potassium phosphate. Only about 30% of the optimal activity can be measured in 5 mM potassium phosphate. Increasing ionic strength also reduces the degree of substrate inhibition and shifts the maximal activity to higher substrate concentrations (Fig. 4).
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100 mM salt. It is not specific for phosphate buffer because the same protection is gained with salts such as KCl and NaCl.
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30 µM. This compares to 2–4 µM for E. coli PGDH. E. coli PGDH also shows positive cooperativity for serine inhibition with a Hill coefficient of 2.0. Unlike the E. coli enzyme, when assayed in phosphate buffer, M. tuberculosis PGDH displays a Hill coefficient of 1.0, indicating the absence of cooperativity for serine inhibition. However, positive cooperativity can be induced by the presence of chloride ions. Table II demonstrates that when chloride ion is present in the phosphate buffer, Hill coefficients greater than 1.0 are observed. Although the effect of chloride on the Hill coefficient seems to be optimal at high chloride ion concentrations (250 mM), the effect is present at physiological levels of chloride ion as well (0.02–0.10 mM). This effect does not involve the potassium ion because it is present in the phosphate buffer salts, and NaCl produces similar results. When chloride ion is missing, even at high salt concentrations such as 250 mM sodium acetate, the effect is not observed. The I0.5, the concentration of inhibitor at which half-maximal inhibition is observed, also increases with the chloride ion concentration. So, although chloride induces cooperativity, it decreases sensitivity to serine. Table II also demonstrates that the effect is maximal for chloride ion among the halogen group. Fluoride ion does not induce cooperativity, whereas bromide and iodide display a small effect. In addition, tartrate ions, which were identified in the crystal structure at an anionic binding site between the intervening and regulatory domains, do not induce cooperativity (Table II).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoglutarate as a substrate. However, the M. tuberculosis enzyme does not display the affinity for 5'-AMP-Sepharose that the rat liver and E. coli enzyme do, although all three utilize NAD as a cofactor. A possible reason for this is addressed in the accompanying article (21), which describes the crystal structure of M. tuberculosis PGDH. The single property that M. tuberculosis PGDH shares with the E. coli enzyme, other than utilizing their common physiological substrates, is that it is inhibited by L-serine at relatively low levels. Interestingly, in potassium phosphate buffer alone, M. tuberculosis PGDH is not inhibited in a positively cooperative manner by L-serine, as is E. coli PGDH. However, the presence of chloride ion converts the inhibition by L-serine from a noncooperative to a cooperative process. Chloride ion is not a requirement for the positively cooperative inhibition of E. coli PGDH by L-serine. Moreover, this modulation occurs at relatively low levels of chloride ion, certainly within physiological levels. As the chloride ion is increased, the I0.5 for L-serine increases, but the value for the Hill coefficient remains >1. Thus, the effect of chloride seems to be fully in force at a relatively low level. The effect of the chloride ion is also manifest in the binding of L-serine to the enzyme, which shows positive cooperativity in the binding of the first two serine molecules in the presence of chloride ion but not in the absence. This is consistent with the mechanism of serine inhibition in E. coli, where it was shown that inhibition of activity is dependent on only the first two serines to bind. It is not known at this point how the chloride ion contributes to this phenomenon.
Modulation of enzyme activity by chloride ion has been reported in a number of cases. For example, it has been reported that rat liver glucose-6-phosphatase (22) and glycogen synthase (23) are inhibited by chloride ion. On the other hand, human cathepsin C (24) is activated by chloride ion. Chloride ion also produces a minor heterotropic effect on hemoglobin, but it does not alter the hill coefficient for ligand binding (25). Thus, the observation that the cooperativity of inhibition is modulated specifically by chloride ion appears to be relatively unique. The crystal structure of M. tuberculosis PGDH does not reveal the site for chloride binding. A potential site for the binding of anionic molecules has been identified by virtue of the binding of tartrate molecules from the crystallization buffer at a site between the intervening and regulatory domains (21). However, tartrate itself does not exhibit an effect on the cooperativity of inhibition by serine.
Fig. 2 presents a sequence alignment of PGDH from eight species. Four are from prokaryotic sources, and four are from eukaryotic sources, two of which are mammalian. Five of these species contain the large C-terminal extension, but only two are eukaryotic organisms. The two other eukaryotes are missing the C-terminal extension, and three prokaryotes contain it. There are 53 residues or 15% that are identical in the N-terminal portion of all the species shown (up to position 346 in Fig. 2). These include residues known to be involved in cofactor binding and at the active site. After position 346, there is less than 1% identity in all eight sequences and less than 2% identity in the C-terminal extensions found in five of the sequences.
Of the PGDH enzymes reported to be inhibited by L-serine, their sensitivity to serine ranges over 3 orders of magnitude. The concentration of L-serine that produces half-maximal inhibition of catalytic activity for PGDH from E. coli, M. tuberculosis, C. glutamicum, and B. subtilis are 3, 30, 640, and 10,000 µM. Some insight into the structural basis for serine sensitivity among these species may be gained from inspection of the homology based on what is known about the E. coli enzyme, although it is not entirely straightforward. In E. coli PGDH, L-serine binds to the allosteric site through hydrogen bonding interaction with a histidine and two asparagine residues. These are found at positions 493, 495, and 513 in Fig. 2. In addition, a potential molecular hinge, defined by a Gly-Gly sequence at positions 356 and 357 in E. coli PGDH, seems to be required for optimal serine inhibition. It has been shown that the Gly-Gly sequence can be replaced by an Ala-Ala sequence without a major effect (16). However, residues with larger side chains interfere significantly with serine-induced inhibition. PGDH from Leishmania and yeast possess the requisite serine-binding residues but are missing the Gly-Gly hinge region. Although not yet tested, they would be predicted to be insensitive to L-serine. On the other hand, PGDH from M. tuberculosis and C. glutamicum possess a Gly-Gly sequence in this area, but their serine-binding residues differ at two positions. However, these residues are capable of forming hydrogen bonds and may function similarly. Both mammalian enzymes are completely devoid of any of these necessary features. The rat liver PGDH has been shown to be completely insensitive to L-serine. A similar situation would be predicted for the human enzyme because it is 94% identical. However, several things are left unexplained by this simple analysis. The C. glutamicum and M. tuberculosis enzymes both have the same residues corresponding to the serine binding positions as well as a Gly-Gly sequence in the right general area, but the C. glutamicum enzyme has been reported to be over a 100-fold less sensitive to L-serine. On the other hand, the B. subtilis enzyme, which has very similar serine-binding residues (but with an Asp replacing an Asn), does not contain a recognizable hinge in the region of position 356. When comparing the enzymes with the C-terminal extension to that of E. coli, the effect of the additional polypeptide must be taken into account and may change the manner in which serine interacts and inhibits the enzyme quite significantly.
The structural and kinetic characteristics of the M. tuberculosis enzyme indicate that it is more closely related to the mammalian PGDHs. However, its sensitivity to serine inhibition suggests that it possesses metabolic features more consistent with the E. coli enzyme. These observations suggest that the M. tuberculosis PGDH may be a link between the E. coli and mammalian enzymes. This is supported by the phylogenetic tree presented by Ali et al. (15).
The intracellular soluble levels of L-serine in E. coli and B. subtilis have been reported to be in the 0.5–1.5 mM range (9). If a similar L-serine concentration is found in M. tuberculosis, the I0.5 for L-serine for M. tuberculosis PGDH suggests that it may well be regulated in vivo by L-serine pools. L-Serine is a central cellular intermediate in bacteria that serves as a source for the synthesis of protein, glycine, cysteine, tryptophan, phospholipids, purines, and single carbon metabolites. In fact, 15% of glycolytic flux is directed into the L-serine biosynthetic pathway (26). In E. coli, PGDH is a constitutive enzyme; that is it cannot grow without an external source of L-serine (27). The gene that codes for M. tuberculosis PGDH, SerA1, has been shown to be an essential gene in that organism (28). Thus, the possibility exists that analogs of L-serine that cannot be used otherwise by the M. tuberculosis organism could be potential new drugs for the control of active M. tuberculosis infection when it is growing mainly on carbon as a source.
The mechanism of E. coli PGDH has been studied in great detail. A recent report has shown that the enzyme displays half-of-the-sites activity for both catalysis and regulation by L-serine (17). Furthermore, that investigation has shown that the magnitude of inhibition of activity depends on the intersubunit relationship of effector site to active site and that this relationship can begin to be defined in a quantitative manner. This picture of how the various ligand sites in PGDH interact has more clearly defined the intermolecular mechanism of allosteric regulation in this enzyme and presented the question as to how general the mechanism may be, not only across species within homologous proteins, but as a general regulatory mechanism for enzymes of diverse function that contain the ACT domain. The unique properties of M. tuberculosis PGDH provide an intriguing and potentially enlightening opportunity to explore this question in regard to the former case and will contribute to our understanding of protein regulatory mechanisms.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3367; Fax: 314-362-4698; E-mail: ggrant{at}molecool.wustl.edu.
1 The abbreviations used are: PGDH, D-3-phosphoglycerate dehydrogenase; HPAP, hydroxypyruvic acid phosphate.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 200 mM KPO4, pH 7.5.
, 1/10 dilution in 20 mM potassium phosphate buffer, pH 7.5; 
, 1/100 dilution in 20 mM potassium phosphate buffer, pH 7.5; 
, 1/500 dilution in 20 mM potassium phosphate buffer, pH 7.5. The amount of enzyme present in the assay cuvette was kept constant (
