The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
Cinnamomum camphora/genetics* ; Cloning, Molecular ; Genes, Plant ; Phosphotransferases (Phosphate Group Acceptor)/genetics* ; Phylogeny ; Sequence Alignment

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

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.
Carbon-Nitrogen Ligases ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Glutamates ; biosynthesis ; Ligases ; Phosphotransferases (Phosphate Group Acceptor) ; genetics

OBJECTIVETo study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis.

METHODSThe wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope.

RESULTSThe survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs.

CONCLUSIONSppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

Brain ; cytology ; Cells, Cultured ; Cytoskeleton ; Endothelial Cells ; cytology ; microbiology ; Escherichia coli ; genetics ; physiology ; Escherichia coli Proteins ; genetics ; Gene Deletion ; Humans ; Phosphotransferases (Phosphate Group Acceptor) ; genetics

Polythioesters newly emerged as a type of novel polymer and they have showed great potential for application in industries. In this study, genes of butyrate kinase (buk) and phosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and poly (3-hydroxybutyrate) (PHB) synthase gene from Thiocapsa pfennigii were used for construction of a metabolic pathway to synthesize the polythioesters. When 3-mercaptopropionate and 3-hydroxybutyrate were fed, poly (3-mercaptopropoinate) [poly (3MP)] and poly(3-mercaptopropionate-co-3-hydroxybutyrate) [poly(3MP-co-3HB)] were synthesized by recombinant Escherichia coli JM109 (pBPP1) harboring the constructed metabolic pathway. Results indicated clearly that all these genes are necessary for the synthesis of poly(3MP) and poly(3MP-co-3HB).
3-Hydroxybutyric Acid ; chemistry ; 3-Mercaptopropionic Acid ; chemistry ; Acyltransferases ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Models, Biological ; Molecular Weight ; Phosphate Acetyltransferase ; genetics ; metabolism ; Phosphotransferases (Carboxyl Group Acceptor) ; genetics ; metabolism ; Plasmids ; Polymers ; chemistry ; metabolism ; Spectrophotometry, Infrared