Neurodegeneration with brain iron accumulation (NBIA) is a disorder characterized by various mixtures of extrapyramidal, pyramidal or psychiatric abnormalities associated with iron accumulation in the basal ganglia. The mutations in the pantothenate kinase gene (PANK2) were found in approximately two thirds of the patients with NBIA. We report three patients wtih NBIA, and two of them showed mutations in the PANK2 gene.
Basal Ganglia ; Brain ; Humans ; Iron ; Iron Metabolism Disorders ; Neuroaxonal Dystrophies ; Phosphotransferases ; Phosphotransferases (Alcohol Group Acceptor)

We investigated the effect of adding intermediate metabolites on cell growth and succinate production. The yield of succinic acid achieved to the highest when 0.5 g/L phosphoenolpyruvic acid (PEP) was added. According to the metabolic network of Actinobaccilus succinogenes NJ113, the metabolic flux was calculated by metabolic flux analysis. The ratio of hexose monophosphate pathway to glycolytic pathway increased from 39.4:60.3 to 76.8:22.6 after adding 0.5 g/L PEP, thus the reducing power was better balanced. The flux of PEP to oxaloacetate was 23.8% higher, which made the succinic acid flux improve from 99.8 mmol/(g DCW x h) to 124.4 mmol/(g DCW x h) and the flux of acetic acid and formic acid decreased by 22.9% and 15.4%, respectively. The key enzyme activity analysis showed that the specific activity of PEP carboxykinase reached to 1910 U/mg with 0.5 g/L PEP addition, which was 74.7% higher than the control; and the specific activity of pyruvate kinase decreased by 67.5%. Finally, the concentration of succinic acid was 29.1 g/L with the yield of 76.2%.
Actinobacillus ; metabolism ; Anaerobiosis ; Culture Media ; Culture Techniques ; methods ; Fermentation ; Phosphotransferases (Paired Acceptors) ; metabolism ; Succinic Acid ; metabolism

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg²⁺), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.
Sphingobacterium/metabolism* ; Phosphotransferases (Phosphate Group Acceptor)/metabolism* ; Amino Acids ; Adenosine Triphosphate ; Regeneration ; Polyphosphates/metabolism*

Bacterial Proteins/metabolism* ; Operon/genetics* ; Phosphotransferases/metabolism* ; Quorum Sensing/genetics* ; Vibrio cholerae/genetics*

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
Cytidine Monophosphate ; metabolism ; Escherichia coli ; genetics ; Industrial Microbiology ; methods ; Phosphotransferases (Phosphate Group Acceptor) ; metabolism ; Uridine Kinase

To understand the effects of sugar whose uptake is dependent or independent on the phosphotransferase system (PTS), two-stage culture of Escherichia coli strain NZN111 that was constructed by disruption of IdhA and pflB encoding the fermentative lactate dehydrogenase (LDH) and pyruvate: formate lyase (PFL) of E. coli W1485, was carried out for organic acids production. When NZN111 was aerobically cultured on fructose (PTS dependent) or maltose (PTS independent), it fermented glucose with succinic acid and pyruvic acid as the major products in subsequent anaerobic culture. The experiments were also performed in a 5-L fermentor. The yields of succinic acid by the fructose-and maltose-grown NZN111 were 0.84 and 0.75 mol/mol, whereas the yields of pyruvic acid were 0.65 and 0.83 mol/mol, respectively. The final ratio of succinic acid to pyruvic acid in the anaerobic stage reached 1.73:1 and 1.21:1, respectively. The different behaviors in anaerobic fermentation by the fructose-, maltose- and glucose-grown NZN111 were likely caused by the regulation of catabolite repression in the aerobic culture stage.
Aerobiosis ; Anaerobiosis ; Carbon ; metabolism ; Escherichia coli ; classification ; metabolism ; Fermentation ; Fructose ; metabolism ; Maltose ; metabolism ; Phosphotransferases ; metabolism ; Pyruvic Acid ; metabolism ; Succinic Acid ; metabolism

AIMTo establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples.

METHODSThe ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products.

RESULTSSPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells.

CONCLUSIONMethods for determining the activity of SPK and the content of SPK in biological samples were established.

Cell Line ; Cytophotometry ; Humans ; Isotope Labeling ; Lysophospholipids ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sphingosine ; analogs & derivatives ; metabolism

Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.
Alkyl and Aryl Transferases ; genetics ; Antimalarials ; metabolism ; Artemisinins ; metabolism ; Enterococcus faecalis ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; methods ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sesquiterpenes ; metabolism ; Transformation, Bacterial

OBJECTIVEDiabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide and its prevalence continues to increase. Currently, therapies for DN provide only partial renoprotection; hence new targets for therapeutic intervention need to be identified. In this review, we summarized the new target, sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway, explored its potential therapeutic role in the prevention and treatment of DN.

DATA SOURCESMost relevant articles were mainly identified by searching PubMed in English.

STUDY SELECTIONMainly original articles and critical review articles by major pioneer investigators in this field were selected to be reviewed.

RESULTSSphK1/S1P pathway can be activated by hyperglycemia, advanced glycation end products, and many pro-inflammatory cytokines, which leads to fibronectin, transforming growth factor-β1 up-regulation and AP-1 activation. And then it could promote glomerular mesangial cells proliferation and extracellular matrix accumulation, mediating the initiation and progression of diabetic renal fibrosis.

CONCLUSIONSSphK1/S1P pathway is closely correlated with the pathogenesis of DN. The results suggest that SphK1/S1P pathway as a new target for clinically improving DN in future is of great prospect.

Diabetic Nephropathies ; enzymology ; metabolism ; Extracellular Matrix ; metabolism ; Humans ; Lysophospholipids ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Signal Transduction ; Sphingosine ; analogs & derivatives ; metabolism

NAD kinase catalyzes the phosphorylation of coenzyme I [NAD(H)] to form coenzyme II [NADP(H)], and NADPH is an important cofactor in L-isoleucine biosynthesis. In order to improve NADPH supply, ppnK, the gene encoding NAD kinase in Corynebacterium glutamicum was cloned and separately expressed in an L-isoleucine synthetic strain, Brevibacterium lactofermentum JHI3-156, by an inducible expression vector pDXW-8 and a constitutive expression vector pDXW-9. Compared with the control strain JHI3-156/pDXW-8, NAD kinase activity of the inducible ppnK-expressing strain JHI3-156/pDXW-8-ppnK was increased by 83.5%. NADP(H)/NAD(H) ratio was also increased by 63.8%. L-isoleucine biosynthesis was improved by 82.9%. Compared with the control strain JHI3-156/pDXW-9, NAD kinase activity of the constitutive ppnK-expressing strain JHI3-156/pDXW-9-ppnK was increased by 220%. NADP(H)/ NAD(H) ratio and NADPH concentration were increased by 134% and 21.7%, respectively. L-isoleucine biosynthesis was increased by 41.7%. These results demonstrate that NAD kinase can improve the coenzyme II supply and L-isoleucine biosynthesis, which would also be useful for biosynthesis of other amino acids.
Brevibacterium ; genetics ; metabolism ; Cloning, Molecular ; Corynebacterium glutamicum ; enzymology ; Isoleucine ; biosynthesis ; Metabolic Engineering ; NAD ; metabolism ; NADP ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism