Dae-Sil Lee

Antibacterial activity of [10]-gingerol and [12]-gingerol isolated from ginger rhizome against periodontal bacteria

Ginger (Zingiber officinale Roscoe) has been used widely as a food spice and an herbal medicine. In particular, its gingerol‐related components have been reported to possess antimicrobial and antifungal properties, as well as several pharmaceutical properties. However, the effective ginger constituents that inhibit the growth of oral bacteria associated with periodontitis in the human oral cavity have not been elucidated. This study revealed that the ethanol and n‐hexane extracts of ginger exhibited antibacterial activities against three anaerobic Gram‐negative bacteria, Porphyromonas gingivalis ATCC 53978, Porphyromonas endodontalis ATCC 35406 and Prevotella intermedia ATCC 25611, causing periodontal diseases. Thereafter, five ginger constituents were isolated by a preparative high‐performance liquid chromatographic method from the active silica‐gel column chromatography fractions, elucidated their structures by nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry and their antibacterial activity evaluated. In conclusion, two highly alkylated gingerols, [10]‐gingerol and [12]‐gingerol effectively inhibited the growth of these oral pathogens at a minimum inhibitory concentration (MIC) range of 6–30 µg/mL. These ginger compounds also killed the oral pathogens at a minimum bactericidal concentration (MBC) range of 4–20 µg/mL, but not the other ginger compounds 5‐acetoxy‐[6]‐gingerol, 3,5‐diacetoxy‐[6]‐gingerdiol and galanolactone. Copyright

Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus

Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of α,α-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40°C than at 80°C and was independent of the substrate concentration. The maximum yield of α,α-trehalose from 10 mM maltose reached 86% at 40°C. In addition, α,β-trehalose was also formed from maltose or α,α-trehalose at 3.5% yield at 80°C.

Crystal structure of the apo form of D‐alanine: D‐alanine ligase (Ddl) from Thermus caldophilus: A basis for the substrate‐induced conformational changes

Introduction. D-alanine:D-alanine ligase (Ddl) catalyses the dimerization of D-alanine before its incorporation in peptidoglycan precursors. The synthesis of D-alanine:Dalanine begins with an attack on the first D-alanine by the -phosphate of adenosine triphosphate (ATP) to yield an acylphosphate. That is followed by attack by the amino group of the second D-alanine, which eliminates the phosphate and produces the D-alanine: D-alanine dipeptide. Peptidoglycan biosynthesis has long been an attractive target for antibacterial drugs, such as D-cycloserine, glycopeptide antibiotics (vancomycin and teicoplanins), and -lactams (penicillin and cephalosporins). Vancomycintype antibiotics, for example, bind directly to the D-alanine: D-alanine terminus, thereby inhibiting crosslinking by the transpeptidase. Notably, bacteria that show vancomycin resistance, which develops after prolonged clinical treatment with vancomycin, possess an inactive Ddl and rely on another ligase, D-alanine:D-lactate ligase (Van), which produces D-alanine:D-lactate rather than D-alanine:Dalanine for cell wall synthesis. The switch from D-alanine: D-alanine peptidoglycan termini to D-alanine:D-lactate results in the loss of crucial hydrogen bonding interactions that causes a 1000-fold reduction in vancomycin binding affinity. X-ray crystallographic studies of Ddl and Van have contributed significantly to our understanding of the ligand specificity these two enzymes and suggest that a His residue in Van plays a critical role. A positive charge on the side chain imidazole nitrogen of His would attract the negatively charged lactate to the second substrate binding site at pH values less than 7, but at higher pH values Van would predominantly synthesize D-alanine:D-alanine. In Ddl, a Tyr residue [Tyr216 in Escherichia coli (Eco) DdlB, Tyr232 in Thermus caldophilus (Tca) Ddl] occupies the same spatial position as the His residue, and the hydroxyl group of the Tyr interacts with the COOH-terminal of the second D-alanine substrate. The structure of Eco DdlB complexed with ADP/ phosphorylated phosphinate (PDB ID: 2DLN) or with ADP/phosphorylated phosphonate (PDB ID: 1IOV) has been determined, as have the structures of Leuconostoc mesenteroides (Lme) D-Alanine:D-Lactate ligase complexed with ADP and phosphinophosphate (PDB ID:1EHI) and Enterococcus faecium (Efa) VanA complexed with ADP and phosphinophosphate (PDB ID:1E4E). However, to analyze the reaction mechanisms of these enzymes and their associated conformational changes, it is necessary to know the structures of both the substrate-bound and substrate-free forms of these enzymes. Our aim in the present study, therefore, was to grow crystals of Ddl that diffracted to high resolution in the absence of substrates. Here we report the X-ray structure of TcaDdl resolved to a resolution of 1.9 A and describe the conformational differences of the apo structure, comparing it with the structures of the previously described transition state analogue complex.

Biochemical characterization of a UDP‐sugar pyrophosphorylase from Thermus caldophilus GK24

An extremely thermostable UDP‐GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion‐exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8‐fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel‐filtration chromatography. The activity was maximum at 86 °C and its half‐life at 95 °C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP‐GlcNAc pyrophosphorylase activity could be enhanced by fructose 1‐phosphate, a precursor of UDP‐GlcNAc. The enzyme showed a broad substrate specificity with sugar 1‐phosphates, including glucose 1‐phosphate, GlcNAc‐1‐P and xylose 1‐phosphate. The enzyme was therefore named UDP‐sugar pyrophosphorylase. The N‐terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N‐terminal sequence is similar to that of UDP‐GlcNAc and UDP‐glucose pyrophosphorylases from other bacterial sources.

Biotransformation of uridine monophosphate (UMP) and glucose to uridine diphosphate-glucose (UDPG) byCandida saitoana KCTC7249 cells

The present study investigates the biotransformation of glucose with uridine monophosphate (UMP) to obtain sugar nucleotide, UDP-glucose (UDPG), by the dried cells ofCandida saitoana KCTC7249. The biotransformation was optimized by varying the concentrations of substrates and phosphate ion. UDPG (24 mM) was biotransformed from 200 mM glucose and 37.5 mM UMP by dried cells of C.saitoana. The glucose yields about 64% UDP-glucose, based on UMP concentration. The addition of glucose-1-phosphate to the reaction mixture accelerated the formation of UDPG from a concentration of UMP. The structure of UDP-glucose obtained was determined with13C NMR and FAB mass spectra. These results indicate that the yeast-dried cells could be used for the production of nucleotide sugars for donor molecules of complex carbohydrate synthesis.

Isolation and purification of methyl mercaptan oxidase fromRhodococcus rhodochrous for mercaptan detection

Methyl mercaptan oxidase was successfully induced fromRhodococcus rhodochrous IGTS8 using methyl mercaptan gas and purified to homogeneity for the detection of mecrcaptans. The purification procedure involved DEAE-Sephacel and Superose 12 column chromatography with recovery yields of 85.8 and 83.3%, and a specific activity of 92.7 and 303.4 units/mg-protein, respectively. The molecular weight of purified methyl mercaptan, oxidase was determined to be 64.5 kDa by SDS-PAGE. The extract from gel filtration chromatography oxidizes methyl mercaptan to produce formaldehyde, which can be easily detected by the purpald-coloring method. Optimum temperature for activity was achieved at 60°C. This enzyme was inhibited by both K2SO4 and NaCl at concentration of less than 100 mM and recovered to original activity at concentration of 200 mM. In the presence of methanol, the activity decreased by 33%.

The Major Outer Membrane Protein of a Periodontopathogen Induces IFN-β and IFN-Stimulated Genes in Monocytes via Lipid Raft and TANK-Binding Kinase 1/IFN Regulatory Factor-3

Surface molecules of pathogens play an important role in stimulating host immune responses. Elucidation of the signaling pathways activated by critical surface molecules in host cells provides insight into the molecular pathogenesis resulting from bacteria-host interactions. MspTL is the most abundant outer membrane protein of Treponema lecithinolyticum, which is associated with periodontitis, and induces expression of a variety of proinflammatory factors. Although bacteria and bacterial components like LPS and flagellin are known to induce IFN-β, induction by bacterial surface proteins has not been reported. In the present study, we investigated MspTL-mediated activation of signaling pathways stimulating up-regulation of IFN-β and IFN-stimulated genes in a human monocytic cell line, THP-1 cells, and primary cultured human gingival fibroblasts. MspTL treatment of the cells induced IFN-β and the IFN-stimulated genes IFN-γ-inducible protein-10 (IP-10) and RANTES. A neutralizing anti-IFN-β Ab significantly reduced the expression of IP-10 and RANTES, as well as STAT-1 activation, which was also induced by MspTL. Experiments using specific small interfering RNA showed that MspTL activated TANK-binding kinase 1 (TBK1), but not inducible IκB kinase (IKKi). MspTL also induced dimerization of IFN regulatory factor-3 (IRF-3) and translocation into the nucleus. The lipid rapid-disrupting agents methyl-β-cyclodextrin, nystatin, and filipin inhibited the MspTL internalization and cellular responses, demonstrating that lipid raft activation was a prerequisite for MspTL cellular signaling. Our results demonstrate that MspTL, the major outer protein of T. lecithinolyticum, induced IFN-β expression and subsequent up-regulation of IP-10 and RANTES via TBK1/IRF-3/STAT-1 signaling secondary to lipid raft activation.

Cloning and expression of the gene for xylose isomerase from Thermus flavus AT62 in Escherichia coli

The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Thermus caldophilus

Introduction. Peptidoglycan biosynthesis is initiated with the formation of UDP-N-acetylmuramic acid via the stepwise action of two enzymes, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and UDP-N-acetylenolpyruvylglucosamine reductase (MurB), which has proven to be attractive targets for the development of antimicrobial agents. MurA catalyzes the first stage of the reaction, transferal of the enolpyruvate moiety of phosphoenolpyruvate to the 30-hydroxyl of UDP-N-acetylglucosamine with the release of inorganic phosphate. The resulting intermediate, enolpyruvyl-UDP-N-acetylglucosamine (EP-UDPGlcNAc), then undergoes a reduction catalyzed by MurB, which utilizes one equivalent of nicotinamide adenine dinucleotide phosphate and a solvent-derived proton. The reduced product, UDP-N-acetlymuramic acid, can then serve as the locus of attachment for the peptide portion of the cell wall. The resulting pentapeptide then participates in the crosslinking that gives the cell wall its rigidity. Mur enzymes have been well-studied, and the structures of MurA-G have been solved. Moreover, the structures of MurB have been classified into two types based on the presence or absence of various secondary structural elements and on how these structural elements interact with substrates. For instance, Escherichia coli MurB is classified as Type I and contains a Tyr loop and split babb fold, whereas Staphylococcus aureus MurB is classified as Type II and lacks these secondary elements. Here, we report the crystal structures of Thermus caldophilus MurB, which is also classified as Type II. This is the first report describing the substrate bound structure of a Type II MurB.

Gentiobiose synthesis from glucose using recombinant β-glucosidase fromThermus caldophilus GK24

Recombinant β-glucosidase fromThermus caldophilus GK24 was easily purified partially by a heat treatment procedure, resulting in 8-fold and recovery yield of 80% from crude enzyme. When the β-glucosidase was incubated with a 80% glucose solution (w/w), gentiobiose (β1,6-glucobiose) was the major product in the reaction mixture. The optimal conditions for producing gentiobiose (11% yields of total sugar) were pH 8–9 and 70°C for 72 h.