In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.
Ligands ; Glycosyltransferases/genetics* ; Sterols ; Phylogeny ; Ascomycota ; Liliaceae/chemistry* ; Melanthiaceae ; Diosgenin ; Sugars ; Glucose ; Uridine Diphosphate

Oxidative Phosphorylation ; Acetylglucosamine ; Uridine Diphosphate N-Acetylglucosamine/metabolism*

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.
Fermentation ; Ginsenosides ; Humans ; Panax/genetics* ; Panax notoginseng ; Saccharomyces cerevisiae/genetics* ; Uridine Diphosphate Glucose

Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.
Ginsenosides/metabolism* ; Glucose ; Panax ; Saccharomyces cerevisiae/metabolism* ; Uridine Diphosphate Glucose

We amplified genes encoding UDP-glucose dehydrogenase, ecohasB from Escherichia coli and spyhasB from Streptococcus pyogenes. Both ecohasB and spyhasB were inserted into T7 expression vector pRX2 to construct recombinant plasmids pRXEB and pRXSB, and to express in E. coli BL21(DE3). After nickel column purification of UDP-glucose dehydrogenases, the enzymes were characterized. The optimum reaction condition of spyHasB was at 30 °C and pH 10. The specific activity reached 12.2 U/mg under optimum condition. The optimum reaction condition of ecoHasB was at 30 °C and pH 9. Its specific activity reached 5.55 U/mg under optimum condition. The pmuhasA gene encoding hyaluronic acid synthase was amplified from Pasteurella multocida and ligated with ecohasB and spyhasB to construct the coexpression vectors pBPAEB and pBPASB, respectively. The co-expression vectors were transformed into E. coli BW25113. Hyaluronic acid (HA) was produced by biotransformation and the conditions were optimized. When recombinant strains were used to produce hyaluronic acid, the higher the activity of UDP-glucose dehydrogenase was, the better its stability was, and the higher the HA production could reach. Under the optimal conditions, the yields of HA produced by pBPAEB/BW25113 and pBPASB/BW25113 in shake flasks were 1.52 and 1.70 g/L, respectively, and the production increased more than 2-3 folds as previously reported.
Biotransformation ; Escherichia coli ; enzymology ; Genetic Vectors ; Glucuronosyltransferase ; genetics ; Hyaluronan Synthases ; Hyaluronic Acid ; metabolism ; Pasteurella multocida ; enzymology ; Streptococcus pyogenes ; enzymology ; Uridine Diphosphate Glucose Dehydrogenase ; metabolism

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
Animals ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2C9 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Drug Interactions ; Glucuronosyltransferase ; metabolism ; Humans ; Ligusticum ; chemistry ; Microsomes, Liver ; enzymology ; NADP ; metabolism ; pharmacology ; Pyrazines ; metabolism ; pharmacokinetics ; Rats ; Uridine Diphosphate Glucuronic Acid ; metabolism ; pharmacology

OBJECTIVETo evaluate pathologically the effect of the single or combined application of UDP-glucose, GDNF and memantine on the improvement of white matter injury in neonatal rats with periventricular leukomalacia (PVL) under light and electron microscopy.

METHODSA five-day-old neonatal rat model for PVL was established by ligation of the lateral common carotid artery following 120-minute hypoxia. Rats were randomly divided into six groups (30 rats in each group): sham-operated, PVL, UDP-glucose (UDP-glucose 2000 mg/kg intraperitoneally after PVL), GDNF (GDNF 100 μg/kg intracerebrally after PVL), tmemantine (memantine 20 mg/kg intraperitoneally after PVL), and a combination administration of three drugs (UDP-glucose, GDNF and memantine). The rats were sacrificed 7 or 21 days after PVL for assessment of pathological changes in the white matter under both light and electron microscopy. The number and thickness of the myelin sheath in the white matter were measured under electron microscopy, and both of pathological grading and scoring were undertaken under light microscopy.

RESULTSThere was rare and sparse myelinogenesis with a loose arrangement of nerve fibers in the white matter under electron microscopy in the PVL group at 7 and 21 days after PVL. The number and thickness of the myelin sheath in the PVL group were significantly less than in the sham-operated, UDP-glucose, GDNF, memantine and combination administration groups (P<0.01). The results of pathological grading of white matter under light microscopy showed that all rats in the PVL group manifested either mild injury (38%-50%) or severe injury (50%-62%) at 7 and 21 days after PVL. The majority of rats (50%-88%) in the four drug administration groups had normal white matter at 7 and 21 days after PVL. The pathological scores at 7 and 21 days after PVL in the PVL group were the highest, and they were significantly higher than in the other five groups (P<0.05).

CONCLUSIONSThe single or combined application of UDP-glucose, GDNF and memantine may significantly improve pathological changes in the white matter of rats with PVL. The favorable effect is inferred to be closely correlated with the improvement of brain microenvironment and the enhancement of nerve regeneration promoted by the three drugs.

Animals ; Brain Ischemia ; drug therapy ; pathology ; Cerebral Ventricles ; pathology ; ultrastructure ; Female ; Glial Cell Line-Derived Neurotrophic Factor ; administration & dosage ; therapeutic use ; Humans ; Infant, Newborn ; Leukomalacia, Periventricular ; drug therapy ; Male ; Memantine ; administration & dosage ; therapeutic use ; Microscopy, Electron ; Rats ; Rats, Sprague-Dawley ; Uridine Diphosphate Glucose ; administration & dosage ; therapeutic use

Galactosemia is a group of inherited enzyme deficiencies characterized by increase in the blood galactose levels. This condition may be associated with deficiencies of galactose-1-phosphate uridyl transferase, galactokinase, or uridine diphosphate galactose-4-epimerase. However, the elevated galactose identified by neonatal screening tests has several other possible etiologies, including hepatic hemangioendothelioma, hepatic hemangioma, and patent ductus venosus with hypoplasia of the portal vein. We report a 13-day-old Korean male with hepatic hemangioendothelioma, which was incidentally detected during the evaluation for suspected galactosemia. Laboratory studies revealed that mildly elevated levels of galactose, galactose-1-phosphate and alpha-fetoprotein, at the time of admission, were gradually decreased to the normal range over the 6 months of observation. Ultrasonography showed a well-defined heterogeneous hypoechoic mass in the liver, and magnetic resonance imaging study showed multiple enhanced mass lesions, which was compatible with the diagnosis of a hepatic hemangioendothelioma. Thus, hepatic imaging, especially ultrasonography, should be performed if neonatal screening suggests galactosemia.
alpha-Fetoproteins ; Galactokinase ; Galactose ; Galactosemias ; Galactosephosphates ; Hemangioendothelioma ; Hemangioma ; Humans ; Infant ; Infant, Newborn ; Liver ; Magnetic Resonance Imaging ; Male ; Neonatal Screening ; Portal Vein ; Reference Values ; UDPglucose-Hexose-1-Phosphate Uridylyltransferase ; Uridine Diphosphate ; Vascular Malformations

In plants, UDP-L-rhamnose is one of the major components of cell wall skeleton. Rhamnose synthase plays a key role in rhamnose synthesis which converts UDP-D-glucose into UDP-L-rhamnose in plants. In this study, we isolated the 1058 bp promoter region of the rhamnose synthase gene AtRHM1 from Arabidopsis genome by PCR, and created a series of deletions of AtRHM1 promoter ranging from -931 bp to +127 bp. The full length of the promoter and its deletion derivatives fused with GUS reporter gene were introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation respectively. The GUS staining and GUS enzymatic activity assay showed that the expression of AtRHM1 is induced at transcriptional level by glucose and the regulatory elements involved in the glucose response are located in the region of -931 bp - -752 bp which contains three G-box motifs.
Arabidopsis ; genetics ; Arabidopsis Proteins ; genetics ; Glucosyltransferases ; genetics ; Plants, Genetically Modified ; genetics ; Promoter Regions, Genetic ; Uridine Diphosphate Glucose ; genetics ; metabolism ; Uridine Diphosphate Sugars ; genetics ; metabolism

The study was purposed to develop a novel cryopreserved agent (CPA) for platelets, to investigate the morphology of cryopreserved platelets in different CPA and the CD62P expression on membrane of platelets after stimulating by thrombin, as well as to compare the effect of adding UDP-Gal on preserved efficiency of preservation solutions. A novel cryopreserved agent consisting of 2% DMSO, thrombosol and UDP-Gal was developed on basis of using higher concentration of DMSO. The morphology of chilled platelets was observed by transmission electron microscope and compared with fresh platelets. The expression of CD62P on the membrane of platelets was detected at 0, l, 3 months. The results indicated that the significant effect of cryopreservation on morphology of platelets was found according to percentages of round, dendritic and irregular shapes of cryopreserved platelets. The protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal were better than that of 5% DMSO. Compared with fresh platelets, the expression of CD62P on platelet membrane decreased obviously after cryopreservation, but not observed difference at preservation for 1 month and 3 months, as well as among 3 kinds of different CPA. It is concluded that the protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal on morphology of platelets are similar, but better than that 5% DMSO. The reaction of cryopreserved platelets to thrombin decreases, while the significant difference is not found among these 3 kinds of CPA. The addition of UDP-Gal to cryopreserved agents not show the protective effect on platelets.
Blood Platelets ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; P-Selectin ; biosynthesis ; genetics ; Uridine Diphosphate Galactose ; pharmacology