Mechanism of the glycosylation step catalyzed by human α-galactosidase: a QM/MM metadynamics study.

The enzyme α-galactosidase (α-GAL), a member of glycoside hydrolase family 27, catalyzes the removal of a nonreducing terminal α-galactose residue from polysaccharides, glycolipids, and glycopeptides. α-GAL is believed to have the double displacement retaining reaction mechanism. In this work, the glycosylation step catalyzed by human α-GAL was computationally simulated with quantum mechanics/molecular mechanics metadynamics. Our simulations show that the overall catalytic mechanism follows a D(N)*A(N)-like mechanism, and the transition state has a oxocarbenium ion like character with a partially formed double bond between the ring oxygen and C5' carbon atoms. In addition, the galactosyl ring of the substrate follows a conformational itinerary of (4)C(1) → [E(3)/(4)H(3)](++) → (1)S(3) along the reaction coordinate.

QM/MM study on the catalytic mechanism of heme-containing aliphatic aldoxime dehydratase.

Aliphatic aldoxime dehydratase (Oxd) catalyzes the dehydration of aliphatic aldoximes (R-CH═N-OH) to the corresponding nitriles (R-C≡N). Quantum mechanics/molecular mechanics (QM/MM) calculations are performed to elucidate the catalytic mechanism of the enzyme on the basis of the X-ray crystal structure of the Michaelis complex. On the basis of the calculations, we propose a complete catalytic cycle of Oxd in which the distal histidine (His320) acts as a general acid/base. In the Michaelis complex, the elimination of the hydroxyl group of aldoxime is facilitated by His320 donating a proton to the hydroxyl group in a concerted way, which is the rate-limiting step. The formed intermediate has a ferric heme iron and an unpaired electron on the nitrogen atom of the substrate coupled to a singlet state. The second step is the deprotonation of the ß-hydrogen of the substrate by His320 after the substrate rotates about the Fe-N bond for ∼180° to yield the neutral product. In the meantime, the heme iron goes back to ferrous state by a one-electron transfer from the substrate to the ferric heme iron, and His320 goes back to the protonated state to proceed with the following reaction. The functions of the protein environment and the active site residues are also discussed.

Dexamethasone induces fetal developmental toxicity through affecting the placental glucocorticoid barrier and depressing fetal adrenal function.

This study evaluates the neuroendocrine-interference mechanism underlying dexamethasone-induced developmental toxicity. Pregnant mice were treated with various doses of dexamethasone (0, 0.5, 2.0 and 8.0mg/kg), corticosterone levels in maternal serum, mRNA expressions of maternal and fetal adrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 responsible for cholesterol side chain cleavage (P450scc) and placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD-2) were measured. And the expressions of StAR and P450scc were also measured in cultured primary human fetal adrenocortical cells treated with various concentrations of dexamethasone (0, 1, 10 and 100 µmol/L) for 24h. Mice suffered from intrauterine growth retardation (IUGR) after exposure to dexamethasone. The IUGR rate was augmented to 42.9% and 95.7% in 2.0 and 8.0mg/kg dexamethasone groups, respectively (P<0.01). The level of maternal serum corticosterone in three dexamethasone groups were decreased to 31.8%, 34.8% and 32.9%, respectively (P<0.05 or P<0.01), as compared with the control. Furthermore, the mRNA expressions of maternal and fetal adrenal StAR and P450scc in 8.0mg/kg dexamethasone groups were decreased to 19.3% and 10.8%, 11.0% and 9.9% of that in the corresponding controls, respectively (P<0.05). The mRNA expressions of placental 11ß-HSD-2 were dose-dependently reduced in dexamethasone groups, particularly, the mRNA decreased to 22.2% in 8.0mg/kg dexamethasone group, as compared with the control (P=0.15). No obvious changes of StAR and P450scc in vitro after dexamethasone treatment. These suggest that prenatal dexamethasone exposure induces fetal developmental toxicity. A possible underlying mechanism is that dexamethasone may affect the placental glucocorticoid barrier and depressing fetal adrenal function.

Elucidation of the methyl transfer mechanism catalyzed by chalcone O-methyltransferase: a density functional study.

The mechanism of the methyl transfer catalyzed by chalcone O-methyltransferase has been computationally investigated by employing the hybrid functional B3LYP. Two models are constructed based on the two conformations of the substrate isoliquiritigenin in the X-ray structure. Our calculations show that the overall reaction is divided into two elementary steps: the water-assisted deprotonation of the substrate by His278 as a catalytic base, followed by the methyl transfer from S-adenosyl-L-methionine (SAM) to the substrate. The calculated rate-limiting barriers for the methyl-transfer step indicate that the catalytic reactions are energetically feasible for both conformations adopted by the substrate.

Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations of the thiopurine S-methyltransferase reaction with 6-mercaptopurine.

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/mol, which is in good agreement with the available experimental rate constant data.

Chemical composition and microstructure of uroliths associated with the feeding of high-level cottonseed meal diet to sheep.

The chemical composition and microstructure of five urolith samples (4 bladder stones and one kidney stone) associated with the feeding of high level of cottonseed meal (CSM) diet to Chinese merino fine wool sheep (Junken breed, Xinjiang) were examined by optical microscope, X-ray diffraction, X-ray energy dispersive spectrometry (EDS), scanning electron microscopy (SEM), and infrared spectroscopy analysis. The bladder stone samples appeared yellow or white, small powder and loose mass, and as finely granular under the optical microscope. However, the kidney stone samples from a experimental sheep were found as small brown mass, higher hardness, and as a cracklike structure. Oxygen, phosphorus, potassium and magnesium were found as four major elements in these uroliths by X-ray energy dispersive spectrometry (EDS). Potassium magnesium phosphate (MgKPO(4)) and potassium magnesium phosphate hexahydrate (MgKPO(4)·6H(2)O) were major components in the bladder stones, while less magnesium ammonium phosphate hexahydrate (MgNH(4)PO(4)·6H(2)O) examined by X-ray diffraction and infrared spectroscopy analysis. However, the newly found prismatic crystals, which were rich in magnesium and pyrophosphate, were identified as magnesium pyrophosphate (Mg(2)P(2)O(7)) in the kidney stone. The bladder stone samples appeared irregular mass and balls, cracked under SEM with low magnification, while appeared cracked, irregular layer-like, honeycomb-like or tiny balls under high magnification. The kidney stone samples were observed as cone, irregular block or layered crystal structures.

Ethanol-induced inhibition of fetal hypothalamic-pituitary-adrenal axis due to prenatal overexposure to maternal glucocorticoid in mice.

Prenatal ethanol exposure has been well documented to be one of the etiological factors responsible for intrauterine growth retardation (IUGR). Previous studies have shown that chronic ethanol exposure during pregnancy elevated the basic level of corticosterone in fetus. However, the potential mechanisms behind them are still unclear. The aim of the present study was to investigate the effects of prenatal ethanol exposure on maternal and fetal hypothalamic-pituitary-adrenal (HPA) axis as well as placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD-2), and to clarify the mechanism of ethanol-induced IUGR. Pregnant mice were intragastricly administrated with ethanol at a dose of 6.4 g kg(-1) d(-1) from day 11 to 17 of gestation and parameters representing fetal growth and development were recorded either. The level of corticosterone in maternal serum was determined by ELISA kit. The mRNA expressions of steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage (P450scc) both in maternal and fetal adrenal, and placental 11ß-HSD-2 were detected by real-time quantitative PCR, respectively. The results showed that fetal body weight significantly decreased, and the incidence of IUGR was obviously increased after prenatal ethanol exposure. Maternal serum corticosterone level was elevated, and the expressions of StAR and P450scc were increased in maternal adrenal while decreased in fetal adrenal. The expression of placental 11ß-HSD-2 was significantly reduced. These results suggest that prenatal ethanol exposure induces an inhibition of fetal HPA axis activity and IUGR occurs. The mechanism may be associated with ethanol-induced maternal HPA axis activation and high glucocorticoid condition, which impair the placental barrier, and lead to an overexposure of elevated maternal glucocorticoid to fetus, and eventually result in the inhibition of the fetal HPA axis.

Catalytic mechanism of hydroxynitrile lyase from Hevea brasiliensis: a theoretical investigation.

Density functional theory (DFT) calculations using the hybrid functional B3LYP have been performed to investigate the catalytic mechanism of hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL). This enzyme catalyzes the cleavage of acetone cyanohydrin to hydrocyanic acid plus acetone. Two models (A and B) of the active site consisting of 105 and 155 atoms, respectively, were constructed on the basis of the crystal structure. Good consistency between the two models provides a verification of the proposed mechanism. Our calculations show that the catalytic reaction proceeds via three elementary steps: (1) deprotonation of the OH-Ser80 by His235 and concomitant abstraction of a proton from the substrate hydroxyl by Ser80; (2) the C-C bond cleavage of the acetone cyanohydrin; and (3) protonation of the cleaved cyanide by His235. The cleavage of the C-C bond is the rate-limiting step with the overall free energy barrier of 13.5 kcal/mol for relatively smaller model A (14.9 kcal/mol for a larger model B) in the protein environment, which is in good agreement with experimental rate. The present results give support to the previously proposed general acid/base catalytic mechanism, in which the catalytic triad acts as a general acid/base. Moreover, the calculated results for model C, with the positive charge of Lys236 removed from model A, show that Lys236 with the positive charge plays a vital role in lowering the reaction barrier of the rate-determining and helps in stabilizing the negatively charged CN(-) by forming a hydrogen bond with the substrate, consistent with the experimental analysis.

Growth retardation of fetal rats exposed to nicotine in utero: possible involvement of CYP1A1, CYP2E1, and P-glycoprotein.

To elucidate the possible metabolic mechanism of intrauterine growth retardation induced by nicotine, this study determines the effects of prenatal nicotine exposure on fetal development and cytochrome P4501A1 (CYP1A1), CYP2E1, and P-glycoprotein (Pgp) expression in maternal liver and placenta. Pregnant rats were given 1.0 mg/kg nicotine subcutaneously twice a day from gestational day (GD) 8 to GD 15, 18, or 21. In nicotine-treated groups, fetal developmental parameters including body weight were significantly lower. The activities of CYP1A1 and CYP2E1 in maternal liver microsomes in nicotine-treated groups increased significantly with progressing gestation when compared with the corresponding control, but returned to the level similar to the control in late pregnancy. Nicotine-treated groups induced pathological changes and increased malondialdehyde (MDA) content in the placenta when compared with the control. The gene expressions of CYP1A1 and CYP2E1 in the placenta increased significantly in nicotine-treated groups on GD 15 and GD 18, but returned to the level similar to the corresponding control on GD 21. In nicotine group, there was a decrease of mdr1a expression on GD 15, GD 18, and GD 21, with the most significant decrease on GD 15. In contrast, no significant difference was found in mdr1b mRNA expression between the nicotine-treated animals and the corresponding control. In comparison with the corresponding control, the placental Pgp protein significantly decreased on GD 15 and GD 18. Our results showed that prenatal nicotine exposure resulted in inhibition of fetal growth significantly. The induction of CYP2E1 and CYP1A1 gene expression by nicotine in the maternal liver and placenta may be involved with the observed increase in oxidative stress and lipid peroxidation. The inhibition of the placental Pgp expression by nicotine may also contribute to an increased susceptibility of the fetus to environmental toxins.

Nicotine-induced prenatal overexposure to maternal glucocorticoid and intrauterine growth retardation in rat.

Overexposure to glucocorticoid during fetal development can result in intrauterine growth retardation (IUGR) as well as other diseases after birth. The purpose of this study is to investigate the possibility of glucocorticoid disturbance-mediated nicotine-induced IUGR after chronic prenatal exposure. Nicotine at 1.0mg/kg twice a day was administered subcutaneously to pregnant rats from gestational day (GD) 8 to GD 15 (mid-gestation) or GD 21 (late-gestation). Placental weights and fetal developmental parameters were recorded. Corticosterone levels were determined by radioimmunoassay. The mRNA expressions of adrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD-2) were determined using real-time quantitative RT-PCR. The results showed that prenatal chronic nicotine exposure causes IUGR in rats (P<0.01); in response to nicotine exposure, maternal serum corticosterone levels were elevated at mid- and late-gestations (P<0.05); mRNA expressions of StAR and P450scc increased in maternal adrenals (P<0.05 or 0.01) but decreased in fetal adrenals (P=0.16 or 0.11). Furthermore, the mRNA levels of placental 11 beta-HSD-2 were reduced at mid- and late-gestations (P<0.05). These results suggest that nicotine-induced IUGR is associated with the disturbances of glucocorticoid homeostasis in maternal and fetal rats. A possible underlying mechanism is that long term nicotine administration leads to fetal overexposure to maternal glucocorticoid by the combined effect of increased maternal glucocorticoid level and impaired placental barrier to it, all of which eventually leads to the fetal adrenocortical dysfunction and IUGR.