• Molecules and Cells
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• v.43 no.8
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• pp.694-704
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• 2020

HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.

• Molecules and Cells
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• v.38 no.2
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• pp.180-186
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• 2015

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the ${\alpha}$-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1724-1730
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• 2012

An ${\alpha}$-L-arabinofuranosidase (TmAFase) from Thermotoga maritima MSB8 is a highly thermostable exo-acting hemicellulase that exhibits a relatively higher activity towards arabinan and arabinoxylan, compared with other glycoside hydrolase 51 family enzymes. In the present study, we carried out the enzymatic characterization and structural analysis of TmAFase. Tight domain associations found in TmAFase, such as an inter-domain disulfide bond (Cys306 and Cys476) in each monomer, a novel extended arm (amino acids 374-385) at the dimer interface, and total 12 salt bridges in the hexamer, may account for the thermostability of the enzyme. One of the xylan binding determinants (Trp96) was identified in the active site, and a region of amino acids (374-385) protrudes out forming an obvious wall at the substrate-binding groove to generate a cavity. The altered cavity shape with a strong negative electrostatic distribution is likely related to the unique substrate preference of TmAFase towards branched polymeric substrates.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.6
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• pp.696-701
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• 2002

Optimal conditions for preparing of chitooligosaccharides from chitosan with cellulase was researched by response surface methodo-logy, Penicillium funiculosum derived cellulase was most effective for chitooligosaccharides production as the point of hydrolyzing activity and commercial utility. The result which measures the change of degrading ratio at time course, 10 hr reaction showed a exponential increase and after that time degrading ratio was not changed. The optimal conditions determined by response surface methodology with central composite design of total 26 species were $0.5\%$ of chitosan, 143 U enzyme, 49$^{\circ}C$ of reaction temperature, 13.2 hr of reaction time and pH 3.8. Major chitooligosaccharides produced from chitiosan on optimal conditions were dimer and trimer.

• Korean Journal of Microbiology
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• v.53 no.4
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• pp.297-303
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• 2017

Leucine-responsive regulatory protein (LRP) is a regulatory protein of molecular weight 18.8 kDa and is widely known to regulate many metabolic and functional activities of operons in Escherichia coli. The gene for Lrp from Escherichia coli in pQE system of 6 ${\times}$ His-tagging was expressed and $^3H$-labeled protein, as well as the wild type Lrp, was purified. The crosslink experiments were performed to analyze the quaternary structure of Lrp at high of $5{\mu}M$ and at low concentrations below $0.3{\mu}M$ with cross linkers, such as glutaraldehyde, 1, 2, 3, 4-diepoxy-butane (DEB), and ethylene glycol bis (succinimidyl succinate) (EGS). In the experiments, we found that the Lrp protein can be formed higher conformation states of tetramer, hexamer, octamer, as well as dimeric state when incubated with the above cross linkers.

• Korean Journal of Food Science and Technology
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• v.30 no.2
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• pp.245-252
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• 1998

The chitosanolytic capabilities of cellulases, glucosidases, proteases and commercial enzymes were evaluated, and effective chitosanolytic cellulases from T. viride, T. reesei and Celluclast, a commercial enzyme from T. reesei were characterized. The reaction of cellulase from T. viride, T. reesei and Celluclast was optimal at pH 5. 0 and $45{\sim}55^{\circ}C$. Max. chitosanolytic activities of cellulases from both T. viride and T. reesei were observed at the enzyme/chitosan ratio=0.1 and chitosan concentration=3.0%. For the possible application of commercial Celluclast to chitosan oligosaccharides production, 3%(w/v) chitosan was reacted with 1%(v/v) Celluclast at pH 5.0 and $55^{\circ}C$. The apparent viscosity decreased by 98% within 30 minutes reaction and Max. contents of 50% EtOH solubles were 70% at 15 hrs reaction. Total reducing sugars were also increased with reaction time and maintained approx. 13.5% after 2hrs reaction. In 15 hrs treated chitosan hydrolyzates, various kinds of chitosan oligosaccharides were produced and contents of chitosan hexamer, known for its antitumor activities, were about 8.0%, about 4 times higher values compared with acid hydrolysis method. The results suggested that chitosan oligosaccharides could be produced with low-cost cellulases from T. reesei.

• BMB Reports
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• v.33 no.4
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• pp.326-331
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• 2000

Acidic chitinases from the gizzards of a broiler were purified to homogeneity, using precipitation with $(NH_{4})_{2}SO_{4}$, ion exchanger chromatography, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The enzymes, GAC1 and GAC2, were purified 180- and 194- folds with a recovery of 4.9% and 2.7%, respectively. The molecular mass of GAC1 and GAC2 were 48.2 kDa and 57.8 kDa, respectively. Chromatofocusing resulted in a pI of 3.1 for both enzymes. The purified enzymes were endochitinases that were devoid of ${\beta}-N-acetylglucosaminidase$ and lysozyme activity. Kinetic studies using $[^3H]chitin$ indicate that GAC1 has a $K_m$ and $V_{max}$ of 1.97 mg/ml and 185 mg/mg protein/h, respectively. The GAC2 has a $K_m$ and $V_{max}$ of 0.42 mg/ml and 92.3 mg/mg protein/h, respectively at optimal pH and temperature (pH 5.0 and $60^{\circ}C$). When the pentamer and hexamer of N-acetylglucosamine (GlcNAc) were used as a substrate, the major product by GAC1 was the dimer of GlcNAc with a differential accumulation of the monomer and trimer, depending upon the substrate. However, the GAC2 produced the dimer and trimer in an equal quantity, regardless of the substrate used. The first 9 $NH_2-terminal$ amino acid residues of the purified gizzard chitinase GAC1 and GAC2 shared a 100% homology. The first 25 $NH_2-terminal$ amino acid residues of GAC1 also shared 55-60% homology with animal chitinases and some animal proteins, such as whey protein and oviduct-specific proteins. However, little homology was found with either microbial and plant chitinases, or egg white lysozyme.

• Microbiology and Biotechnology Letters
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• v.45 no.2
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• pp.168-177
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• 2017

Strain A28-5, which can degrade xylan and agar in solid medium, was isolated from a coastal seawater sample collected from Jeju Island, South Korea. This strain was found to be a gram-negative, $Na^+$-requiring bacterial strain with a polar flagellum for motility. Additionally, the strain was tolerant to antibiotics such as ampicillin and thiostrepton. The G+C content of the genome was 43.96% and menaquinone-7 was found to be the predominant quinone. Major fatty acids constituting the cell wall of the strain were $C_{16:1}$ ${\omega}7c/iso-C_{15:0}$ 2-OH (23.32%), $C_{16:0}$ (21.83%), and $C_{18:1}$ ${\omega}7c$ (17.98%). The 16S rRNA gene sequence of the strain showed the highest similarity (98.94%) to that of Catenovulum agarivorans YM01, which was demonstrated by constructing a neighbor-joining phylogenetic tree. A28-5 was identified as a novel species of the genus Catenovulum via DNA-DNA hybridization with Catenovulum agarivorans YM01, and thus was named as Catenovulum jejuensis A28-5. The formation of tetramers and hexamers of xylooligosaccharides and (neo)agarooligosaccharides, respectively, were confirmed by thin-layer chromatography analysis using an enzyme reaction solution containing xylan or agarose with two crude enzymes prepared from the liquid culture of the strain.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.175-182
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• 1998

We investigated the rate of hepatitis G virus infection among 50 patients who were not infected with the hepatitis C virus but showed symptoms of hepatitis. Viral RNA was extracted from the patients' sera and cDNA was synthesized and amplified by RT-PCR (reverse transcription-polymerase chain reaction) using random hexamer and 5 primers (470-20-1-77F, 470-20-1-211R, 470-20-1-211R-biotin, GV57-4512MF, GV57-4657MR). The amplified PCR products were confirmed by electrochemiluminescence (ECL), liquid hybridization (LH) and Southern blotting (SB). Among the 50 PCR products, by means of ECL, we found 4 samples to be positive and 5 samples to be indeterminate. The GV45-89M probe (5'-CYCGCTGRTITGGGGTGTACfGGAAGGC-3') was end-labelled with gamma-$^{32}P$ ATP and used for liquid hybridization with the PCR products. By using liquid hybridization, we detected specific bands from 4 positive sera and also from one indeterminate serum as determined by ECL. An 1.5% agarose gel electrophoresis of the 9 PCR products which were HGV positive or indeterminate as determined by ECL showed a 160bp band from 4 positive and one indeterminate serum. The 5 PCR products proved to be positive when SB was applied with the GV45-89M probe as well as when LH was applied. LH and SB were shown to have higher sensitivity and specificity than ECL. Two cases among 5 positive cases had relatively high SGOT, SGPT, ALP values when compared with other 48 cases. In summary, we confirmed hepatitis G virus infection in 5 cases among 50 Korean patients showing symptoms of viral hepatitis.

• The Journal of Korean Society of Virology
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• v.29 no.1
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• pp.45-53
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• 1999

We examined the hepatitis G virus infections among 227 Koreans who were healthy or were suspected of hepatitis and determined the phylogenetic relationship based on a part of the NS-5 region of 5 positive samples. Viral RNA was extracted from sera and cDNA was synthesized and subsequently amplified by RT-PCR (reverse transcription-polymerase chain reaction) or RT-nested PCR using random hexamer and NS-5 specific primers (470-20-1-77F, 470-20-1-211R, HGVNESTFO, HGVNESTRE). Five positives were found to belong to samples of patients showing symptoms of viral hepatitis. Primers used for PCR or nested PCR were derived from the NS-5 region. On the other hand, no amplification was detected using primers derived from the 5'-NCR (G-146F, G-401R). We performed TA cloning and sequencing of 5 amplified fragments, and their sequences were compared with those of foreign isolates of HGV. The phylogenetic analysis using MegAlign programme of DNAstar has shown that the Korean isolates are clustered on the phylogenetic tree. In summary, we confirmed the hepatitis G virus infection in 5 cases out of 12 patients showing the symptoms of viral hepatitis. The phylogenetic analysis of sequences of 5 amplified fragments showed that their relations to each other were closer than those to the foreign HGV isolates reported.