PGDH Activity Research Articles

Validating the inhibitory effects of d- and l-serine on the enzyme activity of d-3-phosphoglycerate dehydrogenases that are purified from Pseudomonas aeruginosa, Escherichia coli and human colon

BackgroundWe previously demonstrated that the serA gene is associated with bacterial pathogenicity, including bacterial penetration through the Caco-2 cell monolayers, bacterial motility, bacterial adherence, and fly mortality. l-Serine is known to inhibit the d-3-phosphoglycerate dehydrogenase (PGDH) activity of the SerA protein, and it significantly reduced the bacterial pathogenicity as described above. We also demonstrated that in a PGDH assay using crude extracts isolated from overnight cultures of E. coli overexpressing the P. aeruginosa serA gene, l-serine inhibited the PGDH activity of the SerA protein. The basal PGDH activity of the negative control strain was high, presumably due to contamination of unknown proteins in the crude extracts. Therefore, to further confirm the direct inhibition of PGDH activity of P. aeruginosa SerA by l-serine, we purified and characterized the PGDH from P. aeruginosa and compared it with the previously characterized PGDHs from E. coli, and the human colon as controls.ResultsOptimum pH and ionic strength of the purified PGDHs were different depending on the three species; optimal activity of P. aeruginosa PGDH was at pH 7.5 with 50–100 mM Tris–HCl, E. coli PGDH was at pH 8.5 with 100–200 mM Tris–HCl, and human PGDH was at pH 9.0 with 100–200 mM Tris–HCl. The addition of l-serine reduced the activity of PGDH from P. aeruginosa and E. coli, but not the PGDH from human colon. The median inhibitory concentration (IC50) of l-serine was 630 μM for P. aeruginosa and 250 μM for E. coli, while IC50 of d-serine was much higher than that of l-serine; 76 mM in P. aeruginosa PGDH and 45 mM in E. coli PGDH.ConclusionsThese results suggest that l-serine significantly repressed P. aeruginosa pathogenicity through direct inhibition of the PGDH activity, but was not able to inhibit the human PGDH activity. Oral administration of l-serine to compromised hosts might interfere with bacterial translocation and prevent gut-derived sepsis caused by P. aeruginosa through inhibition of the function of the serA gene product.

D-3-Phosphoglycerate Dehydrogenase.

l-Serine is the immediate precursor of d-serine, a major agonist of the N-methyl-d-aspartate (NMDA) receptor. l-Serine is a pivotal amino acid since it serves as a precursor to a large number of essential metabolites besides d-serine. In all non-photosynthetic organisms, including mammals, a major source of l-serine is the phosphorylated pathway of l-serine biosynthesis. The pathway consists of three enzymes, d-3-phosphoglycerate dehydrogenase (PGDH), phosphoserine amino transferase (PSAT), and l-phosphoserine phosphatase (PSP). PGDH catalyzes the first step in the pathway by converting d-3-phosphoglycerate (PGA), an intermediate in glycolysis, to phosphohydroxypyruvate (PHP) concomitant with the reduction of NAD+. In some, but not all organisms, the catalytic activity of PGDH can be regulated by feedback inhibition by l-serine. Three types of PGDH can be distinguished based on their domain structure. Type III PGDHs contain only a nucleotide binding and substrate binding domain. Type II PGDHs contain an additional regulatory domain (ACT domain), and Type I PGDHs contain a fourth domain, termed the ASB domain. There is no consistent pattern of domain content that correlates with organism type, and even when additional domains are present, they are not always functional. PGDH deficiency results in metabolic defects of the nervous system whose systems range from microcephaly at birth, seizures, and psychomotor retardation. Although deficiency of any of the pathway enzymes have similar outcomes, PGDH deficiency is predominant. Dietary or intravenous supplementation with l-serine is effective in controlling seizures but has little effect on psychomotor development. An increase in PGDH levels, due to overexpression, is also associated with a wide array of cancers. In culture, PGDH is required for tumor cell proliferation, but extracellular l-serine is not able to support cell proliferation. This has led to the hypothesis that the pathway is performing some function related to tumor growth other than supplying l-serine. The most well-studied PGDHs are bacterial, primarily from Escherichia coli and Mycobacterium tuberculosis, perhaps because they have been of most interest mechanistically. However, the relatively recent association of PGDH with neuronal defects and human cancers has provoked renewed interest in human PGDH.

Deletion of gene gnd encoding 6-phosphogluconate dehydrogenase promotes L-serine biosynthesis in a genetically engineered strain of Methylobacterium sp. MB200.

To identify potential target genes involved in L-serine biosynthesis in Methylobacterium sp. MB200 and to evaluate the gnd genetically-engineered strains for L-serine production. Five genes that are not associated with the central metabolic pathway but with L-serine biosynthesis were identified from Methylobacterium sp. MB200 mutants. Gene gnd, encoding 6-phosphogluconate dehydrogenase (PGDH), was selected for further evaluation. The gnd deletion mutant showed a 600% increase in D-serine tolerance and an 80% decrease in PGDH activity compared to Methylobacterium sp. MB200. gnd over-expression did not affect D-serine tolerance, whereas it did increase enzyme-activity up to 136%. Additionally, analysis revealed that in Methylobacterium sp. MB200, L-serine inhibited PGDH activity. The deletion of gnd did not affect growth, whereas it did enhance the biosynthesis of L-serine, resulting in a 225% increase in production of L-serine compared to the wild-type. gnd, one of the five genes identified here that is associated with L-serine synthesis, can be developed as a potential candidate for metabolic engineering to promote L-serine synthesis in Methylobacterium sp. MB200.

Reduced Expression of Hydrogen Sulfide–Generating Enzymes Down-Regulates 15-Hydroxyprostaglandin Dehydrogenase in Chorion during Term and Preterm Labor

Chorionic NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) plays a pivotal role in controlling the amount of prostaglandins in the uterus and has been implicated in the process of labor. Prior studies identified hydrogen sulfide-generating enzymes cystathionine-β-synthetase (CBS) and cystathionine-γ-lyase (CSE) in fetal membranes. We investigated whether hydrogen sulfide is involved in the regulation of PGDH expression in the chorion during labor. The chorionic tissues were obtained from pregnant women at preterm in labor and at term in labor or not in labor at term. Levels of CSE and CBS and hydrogen sulfide production rate were down-regulated in term in labor and preterm in labor groups compared with not in labor at term group. The CBS level correlated to PGDH expression in the chorion. Hydrogen sulfide donor NaHS and precursor l-cysteine dose-dependently stimulated PGDH expression and activity in cultured chorionic trophoblasts. The effect of l-cysteine was blocked by CBS inhibitor and CBS siRNA but not by CSE inhibitor and CSE siRNA. Hydrogen sulfide treatment suppressed miR-26b and miR-199a expression in chorionic trophoblasts. miR-26b and miR-199a mimics blocked hydrogen sulfide upregulation of PGDH expression. Our results indicate that hydrogen sulfide plays pivotal roles in maintenance of PGDH expression in the chorion during human pregnancy. Reduced expression of hydrogen sulfide-generating enzymes contributes to an increased amount of prostaglandins in the uterus during labor.

Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet.

The present study investigated the significance of serine biosynthetic genes for salt stress in sugar beet (Beta vulgaris). We isolated a total of four genes, two each encoding D-3-phosphoglycerate dehydrogenase (BvPGDHa and BvPGDHb) and serine hydroxymethyl transferase (BvSHMTa and BvSHMTb). mRNA transcriptional expression for BvPGDHa was significantly enhanced under salt stress conditions in both leaves and roots of sugar beet, whereas it was reduced for BvPGDHb. On the other hand, BvSHMTa was expressed transiently in leaves and roots under salt stress, whereas expression level of BvSHMTb was not altered. PGDH activity was high in storage root. After salt stress, PGDH activity was increased in leaf, petiole, and root. Recombinant proteins were expressed in Escherichia coli. The K m values for 3-phosphoglycerate in PGDHa and PGDHb were 1.38 and 2.92mM, respectively. The findings suggest that BvPGDHa and BvSHMTa play an important role during salt stress in sugar beet.

Enzyme activity highlights the importance of the oxidative pentose phosphate pathway in lipid accumulation and growth of Phaeodactylum tricornutum under CO2 concentration.

BackgroundRising CO2 concentration was reported to increase phytoplankton growth rate as well as lipid productivity. This has raised questions regarding the NADPH supply for high lipid synthesis as well as rapid growth of algal cells.ResultsIn this study, growth, lipid content, photosynthetic performance, the activity, and expression of key enzymes in Calvin cycle and oxidative pentose phosphate pathway (OPPP) were analyzed in the marine diatom Phaeodactylum tricornutum under three different CO2 concentrations (low CO2 (0.015 %), mid CO2 (atmospheric, 0.035 %) and high CO2 (0.15 %)). Both the growth rate and lipid content of P. tricornutum increased significantly under the high CO2 concentration. Enzyme activity and mRNA expression of three Calvin cycle-related enzymes (Rubisco, 3-phosphoglyceric phosphokinase (PGK), phosphoribulokinase (PRK)) were also increased under high CO2 cultivation, which suggested the enhancement of Calvin cycle activity. This may account for the observed rapid growth rate. In addition, high activity and mRNA expression of G6PDH and 6PGDH, which produce NADPH through OPPP, were observed in high CO2 cultured cells. These results indicate OPPP was enhanced and might play an important role in lipid synthesis under high CO2 concentration.ConclusionsThe oxidative pentose phosphate pathway may participate in the lipid accumulation in rapid-growth P. tricornutum cells in high CO2 concentration.

2-Hydroxyglutarate production is necessary for the reaction catalyzed by 3-phosphoglycerate dehydrogenase in Escherichia coli

Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) catalyzes the NAD+-dependent oxidation of D-3-phosphoglycerate (PG), an intermediate of glycolysis, into 3-phosphohydroxypyruvate (PHP), an intermediate of the serine biosynthetic pathway. The equilibrium of the reaction catalyzed by PGDH is strongly directed towards its substrate, PG. PGDH from Escherichia coli possesses the ability to reduce 2-ketoglutarate (KG) into two isomeric forms of 2-hydroxyglutaric acid (HG). The reductase activity of PGDH is considered “unphysiological”. E. coli PGDH has a very high affinity to NADH (Km = 10 nM) and, after binding NADH, undergoes a conformational change that results in the tight binding of NADH. The order of substrate binding in the reverse reaction is: NADH is the first to bind to the enzyme. Given these findings as well as the known concentrations of substrates in the E. coli pool and the Km’s, we propose that the E-NADH enzyme complex preferably binds KG. It prevents binding of PHP and inhibits the reverse reaction. E. coli PGDH catalyzes two consecutive reactions: E-NADH + KG = E-NAD+ + HG and E-NAD+ + PG = E-NADH + PHP. Oxidation of PG occurs at the expense of KG reduction, resulting in the production of HG, which is a necessary product of the PGDH reaction in E. coli. Over-production of HG has been found in human tumor cells and was thought to be caused by mutant forms of isocitrate dehydrogenase (IDH1/2) gained a new ability to reduce KG to HG (D-2-HG). There is contradictory evidence supporting HG production without the mutations in IDH in several tumors and vice versa. A potential role of human PGDH in HG production is discussed. The role of mutant IDH’s may not be in the production of HG but in the consumption of HG produced by other enzymes.

Characterization, modification, and overexpression of 3-phosphoglycerate dehydrogenase in Corynebacterium glutamicum for enhancing l-serine production

The direct fermentative production of l-serine from renewable biomass using Corynebacterium glutamicum is attracting increasing attention. In this study, wild-type C. glutamicum SYPS-062 produced up to 6.65 ± 0.23 g/L l-serine; to further improve l-serine production, the serA gene was cloned, and the C-terminal domain of 3-phosphoglycerate dehydrogenase (PGDH) from this strain was truncated. When expressed in Escherichia coli, the resultant mutein SerAΔ197 showed a specific PGDH activity of 1.092 ± 0.05 U/mg protein, representing a decrease of 25.87 % from that encoded by serA, and was no longer sensitive to high concentrations of l-serine. When serAΔ591 was overexpressed in C. glutamicum SYPS-062, the activity of PGDH in C. glutamicum pJC1-tac-serAΔ591 increased by 47.72 %, and the resultant strain C. glutamicum pJC1-tac-serAΔ591 could accumulate 7.69 ± 0.22 g/L l-serine. Furthermore, when serAΔ591 was overexpressed in C. glutamicum SYPS-062ΔsdaA, the resultant strain could accumulate 8.84 ± 0.23 g/L l-serine at 102 h, and the yield of l-serine on cells (Y p/x) improved by 60 % when compared with that noted in the control. These results demonstrate that l-serine production in C. glutamicum SYPS-062 could be improved by overexpressing a C-terminal truncation of PGDH in combination with other genetic modifications.

The bacterial Entner–Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron–sulfur cluster enzyme 6-phosphogluconate dehydratase

Replacement of the glycolytic pathway of Saccharomyces cerevisiae by a bacterial Entner–Doudoroff pathway (EDP) would result in lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields in the fermentation of sugars. To establish catabolism of glucose via the EDP in S. cerevisiae requires expression of only two additional enzyme activities, 6-phosphogluconate dehydratase (PGDH) and KDPG aldolase. In this work, KDPG aldolase from Escherichia coli could be successfully expressed in the yeast cytosol with very high enzyme activity. Nevertheless, simultaneous expression of KDPG aldolase and a codon optimized PGDH gene of E. coli could not replace glycolysis or the pentose phosphate pathway in growth experiments. It could be shown that this was due to the very low enzyme activity of PGDH. This bacterial enzyme is a [4Fe–4S] iron–sulfur cluster protein. Several attempts to improve the availability of iron–sulfur clusters or iron in the yeast cells, to attract the iron–sulfur cluster assembly machinery to Leu1-PGDH fusion proteins or to localize the PGDH in the mitochondria did not result in improved enzyme activities. From our results we conclude that establishing functional expression of iron–sulfur cluster enzymes will be a major task for the integration of the EDP and other biochemical pathways in yeast.

The role of maslinic acid in the pentose phosphate pathway during growth of gilthead sea bream(Sparus Aurata)

NADPH produced in the pentose phosphate pathway (PPP) plays a central role in the reductive biosynthesis of membrane lipids, as well as in the maintenance of cell integrity; also, it has a key part in the synthesis of protein, the other member element. For that NADPH is involved in the growth processes. Our main objective is to study the effects of maslinic acid (MA) on kinetics and its molecular nature of NADPH-generating systems in the gilthead sea bream (S. aurata) as well as the possible changes of these enzymes related with several feeding conditions; for this, we have studied the kinetic and expression levels of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), both PPP enzymes, in liver and white muscle. MA, a triterpene, stimulates growth, protein turnover rates and hyperplasia in fish. G6PDH and 6PGDH showed hyperbolic kinetics under all experimental conditions. With MA feeding, the specific activity, maximum velocity and catalytic efficiency of both enzymes increased in both tissue. The Michaelis constant changed with MA and fixed diet, and these changes being in relation to the substrate affinity. Moreover, we found that MA increased the protein levels studied, and this behaviour being consistent with the regulation of the number of enzyme molecules. These results show that G6PDH and 6PGDH are two inducible enzymes regulated by MA. Our findings corroborate that MA affect to the activity and expression of G6PDH and 6PGDH, confirming its role as markers of growth.

6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.

15-Hydroxyprostaglandin dehydrogenase suppresses K-RasV12-dependent tumor formation in Nu/Nu mice.

Oncogenic Ras mutations are early genetic events in colorectal cancer that induce cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) biosynthesis. PGE(2), a downstream product of COX-2, promotes cancer progression by modulating proliferation, apoptosis and angiogenesis. 15-hydroxyprostaglandin dehydrogenase (PGDH) degrades PGE(2) and is down-regulated in colorectal cancer, suggesting that PGDH plays a role in regulating PGE(2) levels and that PGDH over-expression could attenuate Ras-mediated tumorigenesis. Lentiviral transduction was used to express GFP (18.GFP), K-Ras(V12) (18.K-Ras(V12)), PGDH (18.PGDH) or both K-Ras(V12) and PGDH (18.K-Ras(V12).PGDH) in nontumorigenic rat intestinal epithelial (IEC-18) cells. 18.K-Ras(V12) cells exhibited increased phosphorylation of MAP kinases and CREB, proliferation rates, COX-2 and microsomal prostaglandin E synthase (mPGES)-1 expression and PGE(2) and PGI(2) levels. 18.PGDH and 18.K-Ras(V12).PGDH cells had 10(4)-fold increases in PGDH activity with decreased PGE(2) and PGI(2) levels, COX-2 and mPGES-1 expression and proliferation rates. 18.GFP, 18.PGDH, and 18.K-Ras(V12).PGDH cells were unable to grow in soft agar media whereas 18.K-Ras(V12) cells exhibited anchorage-independent cell growth. Xenografts of implanted 18.K-Ras(V12) cells in nu/nu mice produced rapid (2 wk) tumors with uniform antibody staining for COX-2 and mPGES-1 throughout the tumor and elevated PGE(2) levels. Xenografts of 18.K-Ras(V12).PGDH cells exhibited delayed (8 wk) tumor formation with negligible COX-2 and mPGES-1 expression and significantly decreased PGE(2) levels. 18.K-Ras(V12).PGDH tumors had decreased staining of the proliferative marker, Ki-67, and a significant increase in apoptosis in the central region of the tumor. Based on these data, we conclude that PGDH expression suppresses K-Ras(V12)-mediated tumorigenesis in intestinal epithelial cells.

Molecular and functional characterization of d-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.

Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1

BackgroundAs the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.ResultsThe crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.ConclusionThe overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.

15-Hydroxyprostaglandin Dehydrogenase Protein Expression in Human Fetal Membranes With and Without Subclinical Inflammation

Prostaglandins play a central role in the stimulation and maintenance of both term and preterm labor. 15-Hydroxyprostaglandin dehydrogenase (PGDH), localized primarily to chorion trophoblasts, is the key enzyme responsible for the metabolism of prostaglandins. In preterm chorion, levels of PGDH protein and activity were lower when compared to term and were further reduced with the presence of infection, but effects of subclinical inflammation and membrane rupture on PGDH expression are not known. Our objectives were (1) to determine the relative expression of PGDH in amnion and chorion and (2) to determine the effect of preterm premature rupture of membranes (PPROM) and (3) subclinical inflammation on PGDH protein expression in preterm fetal membranes. Fetal membranes were collected from women with idiopathic preterm labor. Patients were divided into preterm birth (1) <32 weeks with PPROM (n = 6), (2) <32 weeks with intact membranes (n = 11), (3) >or=32 and <37 weeks with PPROM (n = 10), and (4) >or=32 and <37 weeks with intact membranes (n = 10). Different antibodies were used to detect protein expression and localization of PGDH in amnion and chorion from these patients using both Western blotting and immunohistochemistry. Antibody T (AbT) localized PGDH to chorion trophoblasts, whereas antibody C (AbC) detected immunoreactive (ir) PGDH predominantly in the amnion mesenchyme. By Western blot, AbT showed a stronger 29-kDa ir-PGDH band whereas with AbC, a stronger 55-kDa ir-PGDH signal was detected. 55-kDa ir-PGDH was significantly higher in PPROM amnion, specifically in the <32 weeks group (P < .05) and with PPROM >24 hours (P < .05). No change was detected in the 29-kDa ir-PGDH in either amnion or chorion with gestational age or the presence and absence of PPROM. In addition, neither form of ir-PGDH was altered significantly with or without subclinical inflammation. ir-PGDH is detectable in both chorion trophoblasts and amnion, especially in the mesenchyme; however, the predominant form of the enzyme differs in the 2 tissues. PPROM and subclinical inflammation do not appear to affect the levels of 29-kDa ir-PGDH protein in the fetal membranes. The differential expression of 55-kDa ir-PGDH in preterm amnion with and without PPROM supports the need for a better understanding of the different forms of PGDH.

Biosynthesis and catabolism of prostaglandin F 2(alpha) (PGF 2α) are controlled by progesterone in the rat uterus during pregnancy

Myometrial quiescence is a key factor in all species to accomplish a successful gestation. PGs play a crucial role in mediating parturition events, and their synthesis and metabolism are regulated by cyclooxygenases (COXs) and NAD +-dependent 15-hydroxy-PG dehydrogenase (PGDH), respectively. Progesterone (P 4) is the hormone responsible for maintaining uterine smooth muscle quiescence during pregnancy. In this work, we have studied the effect of P 4 on the activity of COXs and PGDH, the uterine enzymes involved in the biosynthesis and metabolism of prostanoids in the rat. We found that during pregnancy PGF 2α production and also protein levels of COX-1 and COX-2 were decreased. The exogenous administration of P 4 significantly inhibited the uterine production of PGF 2α and also the protein level of COX-2. PGF 2α, metabolism was assessed by PGDH activity, which resulted high during pregnancy and increased as a result of P 4 administration. These results indicate that PGs levels were negatively modulated by P 4, which could be exerting its effect by increasing PGs metabolism through stimulation on PGDH activity and an inhibition on COX and that is a major mechanism for maintain uterine quiescence in pregnancy.

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine.

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.

Differential Changes in 15-Hydroxyprostaglandin Dehydrogenase and Prostaglandin H Synthase (Types I and II) in Human Pregnant Myometrium

Prostaglandins (PGs) play a key role in the onset of labor in many species and regulate uterine contractility and cervical dilatation. Therefore, the regulation of prostaglandin output by PG synthesizing (PGHS-I and PGHS-II) and metabolizing (PGDH) enzymes in the human myometrium may determine uterine activity patterns in human labor both at preterm and at term. We hypothesized that expression of PGHS isozymes and PGDH in myometrium from women at preterm and term labor would change to favor increased uterotonin (PG) production. Myometrial samples were obtained from the lower uterine segment during cesarean section deliveries from women presenting in preterm, no labor; preterm, labor; term, no labor; term, labor. Immunoreactive (ir-) PGHS and PGDH protein was localized using immunohistochemistry, and changes in protein levels were determined by Western blotting. Ir-PGHS-I and PGHS-II proteins were localized only to myocytes. Ir-PGDH was localized to myocytes in all samples of myometrium examined, but using dual immunofluorescence and immunohistochemistry, ir-PGDH was also detected in cells of the connective tissue. Levels of ir-PGHS-I and PGHS-II protein were not significantly different between no labor and labor tissues, either at preterm or at term. There was no significant effect of gestational age on levels of PGDH, PGHS-I, and PGHS-II protein, but there was a significant decrease in ir-PGDH protein levels in myometrium with labor both at preterm and at term. In addition, there was a decrease in PGDH activity in myometrium from women in labor, both at preterm and at term. Therefore, we conclude that PGDH, PGHS-I, and PGHS-II protein localize within the myocytes of the human pregnant myometrium. A decrease in PGDH protein and activity occurs in association with active labor and may contribute to the amount of bioactive PGs available to act within the human pregnant myometrium at that time.

Cortisol/progesterone antagonism in regulation of 15-hydroxysteroid dehydrogenase activity and mRNA levels in human chorion and placental trophoblast cells at term.

PGs mediate parturition events. 15-Hydroxyprostaglandin dehydrogenase (PGDH) catalyzes the first step in the metabolism of PGs to render them inactive. We have reported previously that cortisol (F) decreases PGDH activity and progesterone (P(4)) maintains PGDH in human chorion and placenta at term. To study the interaction of P(4) and F on the regulation of PGDH, we treated chorion and placental trophoblast cells in culture with combinations of F, dexamethasone, P(4), trilostane, and medroxyprogesterone acetate (MPA). Following a 24-h steroid treatment period and 4-h PGF(2alpha) challenge, culture media and cells were collected for measurement of PGF(2alpha) levels and PGDH mRNA by RIA and Northern blotting analysis. F and dexamethasone decreased PGDH activity and mRNA levels. Exogenous P(4) did not significantly alter PGDH activity or mRNA levels; however, MPA significantly stimulated PGDH activity. Trilostane decreased P(4) production by more than 90% and also decreased PGDH activity and expression. Coincubation with P(4) or MPA reversed trilostane inhibition of PGDH, consistent with a stimulatory role for endogenous P(4) on PGDH. MPA significantly reversed F inhibition of PGDH activity and mRNA levels. In the presence of trilostane, P(4) at equimolar concentration to F reversed F inhibition of PGDH mRNA levels. These findings suggest that F may be acting as an endogenous inhibitor of P(4) action in the regulation of PGDH at term.

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha).

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).