• Molecules and Cells
• /
• 제40권4호
• /
• pp.299-306
• /
• 2017

The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens Vibrio vulnificus and Vibrio cholerae. To date, the full-length AphB crystal structure of V. cholerae has been determined and characterized by a tetrameric assembly of AphB consisting of a DNA binding domain and a regulatory domain (RD). Although acidic pH and low oxygen tension might be involved in the activation of AphB, it remains unknown which ligand or stimulus activates AphB at the molecular level. In this study, we determine the crystal structure of the AphB RD from V. vulnificus under aerobic conditions without modification at the conserved cysteine residue of the RD, even in the presence of the oxidizing agent cumene hydroperoxide. A cysteine to serine amino acid residue mutant RD protein further confirmed that the cysteine residue is not involved in sensing oxidative stress in vitro. Interestingly, an unidentified small molecule was observed in the inter-subdomain cavity in the RD when the crystal was incubated with cumene hydroperoxide molecules, suggesting a new ligand-binding site. In addition, we confirmed the role of AphB in acid tolerance by observing an aphB-dependent increase in cadC transcript level when V. vulnificus was exposed to acidic pH. Our study contributes to the understanding of the AphB molecular mechanism in the process of recognizing the host environment.

• 한국식품과학회지
• /
• 제21권3호
• /
• pp.435-440
• /
• 1989

식품의 안전성 평가를 위한 기초자료로서 각종 활성산소 소거제 및 환원제를 사용하여 식품성분간의 상호반응에 의하여 당의 분해로 생성되는 저분자 카르보닐 화합물의 돌연변이원성과 그 억제기구를 검토하였다. 그 결과, 카르보닐화합물의 돌연변이원성은 glyceraldehyde 등의 ${\alpha}-hydroxycarbonyl$ 화합물보다 glyoxal등의 ${\alpha}-dicarbonyl$ 화합물이 큰 것으로 나타났으며, 그 중에서 methyl glyoxal이 가장 큰 것으로 밝혀졌다. 각종 활성산소 소거제 및 환원제의 돌연변이원성 억제작용은 cysteine, glutathione 및 sodium bisulfite의 억제작용이 큰 것으로 나타나 이들 카르보닐화합물의 돌연변이원성은 주로 산화에 의해 생성되는 일중항산소$(^1O_2)$ 및 그 자체의 반응성에 크게 기인하는 것으로 밝혀졌다. 또한, 이들 카르보닐화합물의 돌연변이원성은 첨가된 활성산소 소거제에 의하여 카르보닐화합물의 자동산화로 생성된 일중항산소, 수산라디칼, 과산화수소 및 superoxide anion 등의 소거 및 환원제에 의하여 카르보닐화합물의 반응성의 소실로 인하여 억제되는 것으로 생각된다. 한편, 공시한 각 활성산소 소거제 및 환원제 단독으로는 돌연변이원성이 없는 것으로 나타났다.

• BMB Reports
• /
• 제49권6호
• /
• pp.349-354
• /
• 2016

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.

• Fisheries and Aquatic Sciences
• /
• 제10권2호
• /
• pp.60-67
• /
• 2007

Myostatin (MSTN; also known as GDF8) is a member of the transforming growth factor ${\beta}-superfamily$ of proteins. MSTN negatively regulates mammalian skeletal muscle growth and development by inhibiting myoblast proliferation. Mice and cattle possessing mutant MSTN alleles display a 'double muscling' phenotype characterized by extreme skeletal muscle hypertrophy and/or hyperplasia. We isolated the full-length cDNA of a novel MSTN gene from S. schlegeli muscle tissue and examined its expression pattern in various tissues. The full-length gene (GenBank DQ423474) consists of 1941bp with an open reading frame of 1134 bp, encoding 377 amino acids that show 62-92% amino acid similarity to other vertebrate MSTNs. The predicted protein contains a conserved proteolytic cleavage site (RXRR) and nine conserved cysteine residues at the C terminus. RT-PCR revealed that the unprocessed and prodomain myostatin mRNAs were predominantly present in muscle, with limited expression in other tissues. However, the mature myostatin mRNA was highly expressed in brain and muscle, intermediately expressed in the gills, intestine, heart, and kidney, and weakly expressed in the liver and spleen.

• Journal of Microbiology
• /
• 제35권1호
• /
• pp.72-77
• /
• 1997

The baculovirus gp64 glycoprotein is a major component of the envelope protein of budded virus (BV). It has been shown that the gp64 glycoprotein plays an essential role in the infection process, especialy fusion between virus envelope and cellular endosomic membrane. Recently we reported optimal conditions required for gp64-mediated membrane fusion in pGP64 DNA transfected Spodoptera frugiperda (Sf9) cells (H. J. Kim and J. M. Yang, Jour, Microbiology, 34.7-14). In order to investigate the role of hydrophobicity within the fusion domain of the gp64 glycoprotein for membrane fusion, 13 mutants which have substitution mutation within hydrophobic region I were constructed by PCR-derived site-derected mutagenesis. Each mutated gp64 glycoproteins was transiently expressed by transfecting plasmid DNA into Spodoptera frugiperda (Sf9) cells. Oligomerization of the transisently expressed gp64 glycoproteins was a nalysed by running them on SDS-polyacrylamide gel electrophoresis under non-reducing condition followed by immunoblotting. All of the mutant gp64 glycoproteins expect cysteine-228 were able to form trimers. These results suggest that hydrophobic region I of the gp64 may not be responsible for the oligomerization of the gp64 glycoprotein.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2000년도 추계학술발표대회
• /
• pp.24-31
• /
• 2000

In order to find out SoxR-reducing system in E. coli, we generated Tn10-insertion mutants and screened for constitutive expression of SoxS in a soxS-lacZ fusion strain. One mutation was mapped in rseB, a gene in rseABC (Regulation of SigmaE) operon. The constitutive soxS-expressing phenotype was due to the polar effect on the downstream gene, rseC. RseC is likely to function as a component of SoxR reduction system because SoxR was kept in oxidized form to activate soxS expression in rseC mutant. RseC is an integral membrane protein with an N-terminal cysteine-rich domain in the cytoplasm. The functionally critical cysteines were determined by substitution mutagenesis. The truncated N-terminal domain of RseC reduced the soxS transcription by $50\%$ as judged by in vitro transcription assay. Currently RseC is believed to be a reducing factor for SoxR. However, the mechanism for the reduction needs further investigation.

• Molecules and Cells
• /
• 제27권6호
• /
• pp.673-680
• /
• 2009

Conversion of the normal soluble form of prion protein, PrP ($PrP^C$), to proteinase K-resistant form ($PrP^{Sc}$) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from ${\alpha}$-helix to ${\beta}$-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

• BMB Reports
• /
• 제30권6호
• /
• pp.397-402
• /
• 1997

A thiol-specific spin label was attached to cysteine-102 of yeast cytochrome c and electron paramagnetic resonance (EPR) spectra were measured as a function of added cytochrome c oxidase concentration. The intensity decreased due to line broadening as cytochrome c formed a complex with cytochrome c oxidase and reached a minimum when the ratio of cytochrome c to cytochrome c oxidase became one. Replacement of either Lys-72 or Lys-87 of cytochrome c by Glu did not result in a significant change in binding affinity. Interestingly the K72E mutant, unlike K87E, had a much lower rate of electron transfer than the wild type. These results indicate that many positively charged residues as a group participate in complex formation but Lys-72 might be important for cytochrome c to be locked in an orientation for an efficient electron transfer. A stoichiometry of 1 was also confirmed by optical absorption of the cytochrome c-cytochrome c oxidase complex which had been run through a gel chromatography cloumn to remove unbound cytochrome c. The EPR spectrum of this 1:1 complex, however, was a mixture of two components. This explains a biphasic kinetics for a single binding site on cytochrome c oxidase without invoking conformational transition.

• Journal of Microbiology and Biotechnology
• /
• 제19권12호
• /
• pp.1506-1513
• /
• 2009

$\alpha$-Dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable $\alpha$-dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi $\alpha$-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi $\alpha$-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wild-type dextranase, the mutant DexM2 (D279C/S289C) showed a more than $13^{\circ}C$ improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest $V_{max}$.

• 한국해양바이오학회지
• /
• 제2권4호
• /
• pp.224-229
• /
• 2007

Muscle tissue expresses many muscle-specific genes, including myostatin (also known as GDF8) that is a member of the transforming growth factor-beta superfamily. Myostatin (MSTN) negatively regulates mammalian skeletal muscle growth and development by inhibiting myoblast proliferation. Mice and cattle possessing mutant MSTN alleles display a 'double muscling' phenotype characterized by extreme skeletal muscle hypertrophy and/or hyperplasia. In this study, we first have characterized partial cDNA of a MSTN gene from the muscle tissue in the F. chinensis and examined its expression pattern in various tissues. The partial MSTN gene (GenBank accession number EU 131093) in the F. chinensis was 1134 bp, encoding for 377 amino acids that showed 63-93% amino acid similarity to other vertebrate MSTNs, containing a conserved proteolytic cleavage site (RXRR) and conserved cysteine residues in the C-terminus. Based on a RT-PCR, the MSTN gene was expressed in the all tissues of F. chinensis used in this study.