Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.
Escherichia coli/genetics* ; Transketolase/chemistry* ; Tartrates ; Escherichia coli Proteins/genetics*

PURPOSE: The present study was designed to explore the potential of flunarizine for cisplatin induced painful uremic neuropathy in rats. METHODS: Cisplatin (2 mg/kg; i.p., for 5 consecutive days) was administered and renal uremic markers i.e., serum creatinine were estimated on days 4 and 25. Behavioral changes were assessed in terms of thermal hyperalgesia (hot plate, plantar, tail immersion, and tail flick tests at different time intervals). Biochemical analysis of total calcium, superoxide anion, DNA, and transketolase, and myeloperoxidase activity in tissue samples was also performed. Furthermore, flunarizine (100, 200, and 300 microM/kg; p.o., for 21 consecutive days) was administered to evaluate its potency on uremic neuropathy, and the results were compared with those for the carbamazepine-treated (30 mg/kg; p.o., for 21 consecutive days) groups. RESULTS: Flunarizine attenuated the cisplatin-induced uremic neuropathy, and the degree of behavioral and biochemical changes in serum and tissue samples in a dose dependent manner. The medium and high doses of flunarizine were shown to produce a significant effect on cisplatin induced painful uremic neuropathy. CONCLUSIONS: Our results indicate the potential of flunarizine for anti-oxidative, anti-inflammatory, and neuroprotective actions. Therefore, it may have use as a novel therapeutic agent for the management of painful uremic neuropathy.
Animals ; Calcium ; Cisplatin ; Creatinine ; DNA ; Flunarizine ; Hyperalgesia ; Immersion ; Neurotoxins ; Peroxidase ; Rats ; Superoxides ; Transketolase ; Uremia

Transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, catalyzes several key reactions of non-oxidative branch of pentose phosphate pathway. TK is a homodimer with two active sites that locate at the interface between the contacting monomers. Both ThDP and bivalent cations are strictly needed for TK activation, just like that for all ThDP-dependent enzymes. TK exists in all organisms that have been investigated. Up to now, one TK gene (TKT) and two transketolase-like genes (TKTL1 and TKTL2) have been identified in human genome. TKTL1 is reported to play a pivotal role in carcinogenesis and may have important implications in the nutrition and future treatment of patients with cancer. Researchers have found TK variants and reduced activities of TK enzyme in patients with neurodegenerative diseases, diabetes, and cancer. Recent studies indicated TK as a novel role in the prevention and therapy of these diseases.
Animals ; Humans ; Models, Molecular ; Neurodegenerative Diseases ; enzymology ; Research ; trends ; Transketolase ; chemistry ; genetics ; metabolism

OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.
Cell Cycle ; Maturation-Promoting Factor ; Meiosis ; Metaphase ; Oocytes ; Pentose Phosphate Pathway ; Protein Kinases ; Ribosemonophosphates ; RNA Interference ; RNA, Double-Stranded ; Transketolase

In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.
3-Deoxy-7-Phosphoheptulonate Synthase ; genetics ; Amino Acids, Aromatic ; biosynthesis ; Biosynthetic Pathways ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Metabolic Engineering ; Shikimic Acid ; analogs & derivatives ; metabolism ; Transketolase ; genetics

OBJECTIVE: To investigate the functional mechanism of metadherin (MTDH), hypoxia-inducible factor-1 alpha (HIF-1α) and transketolase-like gene 1 (TKTL1) and their interaction with each other in laryngeal carcinoma development. METHOD: Thirty laryngeal carcinoma samples and 20 samples of para-carcinoma tissue were taken from the patients received operation treatment. The expression levels of MTDH, HIF-1α and TKTL1 were detected in all the samples by SP immunohistochemical methods. The data were analyzed by the SPSS13.0 statistical software. RESULT: The positive expression rate of MTDH, HIF-1α and TKTL1 in the 30 cases of laryngeal carcinoma was 56.67%, 60.00% and 63.33%, respectively, which was 20.00%,10.00% and 15.00% respectively in the para-carcinoma tissue, the difference of the positive expression rate of the tested objects between these two groups was statistically significant (P < 0.05 or P < 0.01). Of the 30 cases of laryngeal carcinoma, the positive expression rate of MTDH, HIF-1α and TKTL1 in the cases with lymphnode metastasis was 84.62%, 84.62% and 79.62%, respectively, compared with the rate in those without lymph nodes metastasis, which was 35.29%, 41.18% and 35.29%. The difference of the positive expression rate of the tested objects between these two groups was statistically significant (P < 0.05). In the tissue of poorly differentiated squamons cell carcinoma verified by pathology, the positive expression rates of MTDH and HIF-1α was 73.68% and 84.21%, respectively, while that in the tissue of well differentiated squamous carcinoma was 27.27% and 18.18%. The difference of the positive expression rate of the tested objects between these two groups was statistically significant (P < 0.05 or (P < 0.01). The positive expression rate of TKTL1 in laryngeal carcinoma at stage I-II was 25.00% and that at stage III-IV was 72.22%, the difference between which was statistically significant (P < 0.05). The positive expression rate of HIF-1α in laryngeal carcinoma at stage I-II was 33.33% and that at stage III-IV was 77. 78%, the difference between which was statistically significant (P < 0.05). The expression of MTDH, HIF-1α and TKTL1 showed no relationship with age,smoking amount or the tumor location (P > 0.05). The Spearman's rank correlation analysis showed that the positive expression rates of MTDH and HIF-1α in laryngeal carcinoma had positive correlation (r = 0.384, P < 0.05); the positive expression rates of TKTL1 and HIF-1α in laryngeal carcinoma had positive correlation (r = 0.508, P < 0.01); But there was no significant correlation between the positive expression rates of MTDH and TKTL1 in laryngeal carcinoma (r = -0.107, P > 0.05). CONCLUSION It suggested that these three proteins may have close relationship with the occurrence, invasion and metastasis of the laryngeal carcinoma. MTDH and TKTL1 may be expected to be new clinical targets for laryngeal carcinoma treatment and it could offer theoretical basis for the prognosis of the laryngeal carcinoma.
Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Cell Adhesion Molecules ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Laryngeal Neoplasms ; diagnosis ; metabolism ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; Transketolase ; metabolism

PURPOSE: The phosphodiesterases (PDEs) are critical components in the cyclic AMP/protein kinase A and the cyclic GMP/phosphokinase G signaling pathways. The cAMP and cGMP pathways are regulated by activation and dissolution of PDEs. Benfotiamine, a lipophilic derivation of thiamine is known an activator of transketolase, is reported to prevent diabetic nephropathy by decreasing proteinuria and reducing oxidative stress. We did this study to investigate the effect of benfotiamine in type 2 diabetic rat kidneys. MATERIALS AND METHODS: We prepared 10 male Long-Evans Tokushima Fatty (LETO: control) and 20 male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which developed non-insulin-dependent diabetes mellitus (NIDDM) naturally. An oral glucose tolerance test confirmed diabetic development in the OLETF rats at 26 weeks. We classified 10 of the OLETF rats into Group I, the no treatment group and the other 10 into Group II, the treatment group. Group II received 100 mg/kg benfotiamine after developing DM. At 44 weeks, we checked kidney weight, serum glucose, free testosterone, insulin, total cholesterol, and triglyceride before sacrifice. We designed the primers for rat PDE5, PDE5A1, and PDE5A2 genes were carried out semiquantitive multiplex RT-PCR. Immunohistochemical staining was performed for monoclonal mouse anti-cGB-PDE5 and mouse monoclonal anti-smooth muscle alpha-actin. RESULTS: For the Control Group, Group I, and Group II, kidney weight was 2.13+/-0.23, 2.08+/-0.22, and 1.94+/-0.44 g; serum glucose was 279.50+/-56.79, 338.00+/-55.00, and 314.71+/-139.1 mg/dl; free testosterone was 1.46+/-1.08, 1.05+/- 0.42, and 0.72+/-0.56 pg/dl; insulin was 1.03+/-0.43, 1.09+/-0.83, and 1.15+/-1.08 ng/ml; total cholesterol was 86.83+/-4.79, 132.00+/-7.69, and 118.14+/-30.93 mg/dl; and triglyceride was 78.83+/-16.47, 177.83+/-75.62, and 194.57+/-92.57 mg/dl, respectively. All three groups expressed PDE5, PDE5A1, PDE5A2 mRNA, but Group I PDE5 mRNA expression was lower than that of Group C, II. However, the expression of PDE5A1 and PDE5A2 mRNA was not significantly different among the three groups. CONCLUSIONS: Serum cholesterol, triglyceride, and glucose were significantly higher in OLETF than in LETO rats. The PDEs were lower in diabetic rat (OLETF) kidneys and PDEs may play a significant role in the development of diabetic renal complications. Benfotiamine is suggested to increase expression of PDE5 mRNA in the type 2 diabetes rat kidney, but the difference in expression levels between PDE5A1 and PDE5A2 was not significant. These findings suggest that benfotiamine may play a specific role in diabetic changes of the rat kidney via a PDE5-related pathway, but it is not clear whether subtype PDE5A1 and PDE5A2 genes play a specific role.
Actins ; Animals ; Cholesterol ; Diabetes Mellitus ; Diabetes Mellitus, Type 2 ; Diabetic Nephropathies ; Diethylstilbestrol ; Glucose ; Glucose Tolerance Test ; Humans ; Insulin ; Kidney ; Male ; Mice ; Muscles ; Oxidative Stress ; Phosphoric Diester Hydrolases ; Phosphotransferases ; Protein Isoforms ; Proteinuria ; Rats ; Rats, Inbred OLETF ; RNA, Messenger ; Testosterone ; Thiamine ; Transketolase

Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Pyruvate Synthase ; genetics ; metabolism ; Transketolase ; genetics ; metabolism