Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.     

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.     

Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.     

Prostaglandin catabolism in fetal and maternal tissues--a study of 15-hydroxyprostaglandin dehydrogenase and delta 13 reductase with specific assay methods

Recent studies have demonstrated that extraductal tissues such as lung are important sources of prostaglandin E2 which maintains the patency of ductus arteriosus in fetuses and prematurely-born infants. Also, organs such as lung are known to be active in the catabolism of PGE2. Earlier studies of enzymes involved in the catabolism of PGE2 such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and delta 13 reductase all used non-specific methods. In the present report, we studied 15-PGDH in fetal and maternal rat lung, kidney, and fetal lamb lung, kidney and ductus arteriosus with the use of a specific substrate (15-S)-[15(3)H-PGE2]. In addition, we measured the activity of delta 13 reductase in these tissues by measuring the conversion of [1-14C]-15-keto PGE2 to [1-14C]-15-keto-13,14-dihydro PGE2. The results from these studies demonstrated that in fetal rat lung and kidney, 15-PGDH activities increased rapidly while delta 13 reductase remained unchanged during late gestation. Ductus arteriosus possessed little 15-PGDH activities. These results strongly suggest that extraductal regulation of PGE2 metabolism is important in determining ductal caliber in fetuses and prematurely delivered neonates.     

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Estradiol -17-beta enhances the formation of (3H)-PGF2 alpha from (3H)-PGE2 in the uterus isolated from ovariectomized rats

Formation of (3H)-PGF2 alpha and (3H)-13, 14,dihydro-15-keto-PGF2 alpha from (3H)-PGE2 by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE2 + NADPH + (3H)-PGE2 + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus. Spayed animals were injected with progesterone (P) or with estradiol-17-beta (Eo) at a dose of 1.0 or 50.0 micrograms. Conversion of (3H)-PGF2 alpha to (3H)-PGE2 or to (3H)-13, 14,dihydro-15-keto-PGF2 alpha did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 micrograms Eo. However, 50.0 micrograms Eo induced a significant conversion after 30 (P less than 0.01) and 60 (P less than 0.001) min of incubation. It is concluded that Eo, at the 50.0 micrograms dose, but not the 1.0 microgram dose of Eo, nor progesterone, stimulated conversion of (3H)-PGE2 into (3H)-PGF2 alpha or (3H)-13, 14,dihydro-15-keto-PGF2 alpha, presumably through the activity of the enzyme PGE2-9-keto-reductase.     

Experimental hyperthyroidism in rats suppresses in vitro prostaglandin metabolism in lung and kidney.

Metabolism of prostaglandin (PG) F2alpha and PGE2 was depressed 40--62% in 100,000 g cytoplasmic supernatants of lungs and kidneys prepared from rats made hyperthyroid by 18 daily L(-) thyroxine injections (200microgram, s--c). These hyperthyroid rats had elevated serum thyroxine levels, cardiac hypertrophy and thyroid atrophy. There were no differences in soluble protein concentrations, NAD+ utilisation by endogenous enzymes and substrates, or in the NAD+ dependence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) between the supernatants prepared from hyperthyroid rats and saline-injected controls. Thyroxine did not inhibit PG metabolism in vitro up to 260 micrometer. These results suggest that thyroxine specifically decreases intracellular levels of PG-metabolising enzymes, especially of the rate-limiting 15-PGDH. Metabolism of PGF2alpha and PGE2 by 15-PGDH was faster in smaller rats and declined with increasing animal weight. These studies imply that some of the clinical features of hyperthyroidism in man might be caused by deficiencies in PG metabolism.     

Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities

Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.     

15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.     

Experimental hyperthyroidism in rats suppresses in vitro prostaglandin metabolism in lung and kidney

Metabolism of prostaglandin (PG) F2α and PGE2 was depressed 40–62% in 100,000 g cytoplasmic supernatants of lungs and kidneys prepared from rats made hyperthyroid by 18 daily L(-) thyroxine injections (200μg,s-c). These hyperthyroid rats had elevated serum thyroxine levels, cardiac hypertrophy and thyroid atrophy. There were no differences in soluble protein concentrations, NAD⁺ utilisation by endogenous enzymes and substrates, or in the NAD⁺ dependence of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) between the supernatants prepared from hyperthyroid rats and saline-injected controls. Thyroxine did not inhibit PG metabolism in vitro up to 260 μM. These results suggest that thyroxine specifically decreases intracellular levels of PG-metabolising enzymes, especially of the rate-limiting 15-PGDH. Metabolism of PGF2α and PGE2 by 15-PGDH was faster in smaller rats and declined with increasing animal weight. These studies imply that some of the clinical features of hyperthyroidism in man might be caused by deficiencies in PG metabolism.     

Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins

Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.     

Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure-function relationships. Using an approach of the combined structure prediction, molecular docking, site-directed mutagenesis, and enzymatic activity assay, we have developed the first three-dimensional (3D) model of the substrate-binding domain (SBD) of mPGES-1 and its binding with substrates prostaglandin H2 (PGH2) and glutathione (GSH). In light of the 3D model, key amino acid residues have been identified for the substrate binding and the obtained experimental activity data have confirmed the computationally determined substrate-enzyme binding mode. Both the computational and experimental results show that Y130 plays a vital role in the binding with PGH2 and, probably, in the catalytic reaction process. R110 and T114 interact intensively with the carboxyl tail of PGH2, whereas Q36 and Q134 only enhance the PGH2-binding affinity. The modeled binding structure indicates that substrate PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the -SH group of GSH. The -SH group of GSH is expected to attack the peroxy group of PGH2, initializing the catalytic reaction transforming PGH2 to prostaglandin E2 (PGE2). The overall agreement between the calculated and experimental results demonstrates that the predicted 3D model could be valuable in future rational design of potent inhibitors of mPGES-1 as the next-generation inflammation-related therapeutic.     

Indomethacin increases 15-PGDH mRNA expression in HL60 cells differentiated by PMA

We previously reported an induction of 15-hydroxyprostaglandin dehydrogenase type I mRNA (15-PGDH) expression accompanied by a decrease in prostaglandin E2(PGE2) levels during cord blood monocytes differentiation into preosteoclastic cells by 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3). These results suggested a role of prostaglandin (PG) enzymes in adhesion and/or differentiation of monocytes.In the present work, we studied modulation of gene expression of PG metabolism enzymes mRNAs in HL60 cells differentiated by phorbol myristate acetate (PMA) into the monocyte/macrophage lineage. We showed that adhesion of HL60 induced by PMA causes an increase of cyclooxygenase 2 (COX 2) and 15-PGDH mRNAs. When adding indomethacin, a non steroidal antiinflammatory drug known to inhibit COX activity, the cells remained attached and expressed large amounts of 15-PGDH mRNA while COX 2 mRNA expression remained unchanged. Indomethacin, in association with PMA can consequently exert a dual control on key enzymes of PGE2 metabolism without modifying adhesion of the cells.     

Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.     

Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation

Background

Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear.

Methodology/Principal Findings

We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential.

Conclusions/Significance

Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.     

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.