• 제목/요약/키워드: Homodimer

검색결과 46건 처리시간 0.025초

마이코박테리아의 adenylyl cyclase (Adenylyl Cyclases in Mycobacteria)

  • 전한승;고인정;오정일
    • 생명과학회지
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    • 제21권3호
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    • pp.473-479
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    • 2011
  • Adenylyl cyclase (AC)는 ATP로부터 cAMP를 형성하는 반응을 촉매한다. AC에 의해 생산된 cAMP는 다양한 신호전달 경로에서 이차전달자로 사용되고 많은 종에서 다양한 세포기능을 조절한다. AC는 1차구조에 따라 6개의 그룹으로 나눌 수 있다. 진핵생물과 Mycobacterium 속에 속하는 세균에서는 class III에 속하는 AC만이 발견된다. Class III에 속하는 AC의 경우 catalytic cyclase 도메인이 dimer를 형성해야만 활성부위가 형성되고 활성을 가지게 된다. 포유류의 AC는 하나의 polypeptide에 2개의 catalytic cyclase 도메인을 가지고 있고, 이 두 개의 도메인이 intramolecular dimerization을 통해서 활성부위를 형성한다. 반면에 mycobacteria의 AC는 polypeptide에 한 개의 catalytic cyclase 도메인을 가지고 있고, homodimer의 4차구조를 형성하여 활성을 가지게 된다. Class III AC의 활성을 위해서 필요한 6개의 아미노산 잔기가 활성부위에 잘 보존되어 있다. 이 6개의 아미노산 잔기는 $Mg^{2+}$과 결합을 하는 2개의 aspartate 잔기쌍, 기질특이성을 부여하는 lysine-aspartate 잔기쌍, 그리고 반응 전이상태를 안정화시키는 arginine-asparagine 잔기쌍들로 이루어져 있다. Mycobacterium tuberculosis H37Rv에서는 16개의 AC 유전자가 발견되었으며, 이 AC의 연구된 특성에 대해 본 총설에서 다룰 것이다.

Cloning, Expression, and Characterization of UDP-glucose Pyrophosphorylase from Sphingomonas chungbukensis DJ77

  • Yoon, Moon-Young;Lee, Kyoung-Jin;Park, Hea-Chul;Park, Sung-Ha;Kim, Sang-Gon;Kim, Sung-Kun;Choi, Jung-Do
    • Bulletin of the Korean Chemical Society
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    • 제30권6호
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    • pp.1360-1364
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    • 2009
  • The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

E. coli 발현 시스템에 의해 생산된 recombinant human bone morphogenetic protein-2의 정제와 생물학적 활성 (Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system)

  • 최경희;문금옥;김수홍;윤정호;장경립;조규성
    • Journal of Periodontal and Implant Science
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    • 제38권1호
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    • pp.41-50
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    • 2008
  • Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

  • Yoon, Moon-Young;Park, Hye-Yeon;Park, Hae-Chul;Park, Sung-Ha;Kim, Sung-Kun;Kim, Young-Chang;Shin, Mal-shik;Choi, Jung-Do
    • Bulletin of the Korean Chemical Society
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    • 제30권7호
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    • pp.1547-1552
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    • 2009
  • Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

Cloning, Expression, and Biochemical Characterization of dTDP-Glucose 4,6-Dehydratase Gene (gerE) from Streptomyces sp. GERI-155

  • Lee, Hei-Chan;Sohng, Jae-Kyung;Kim, Hyung-Jun;Nam, Doo-Hyun;Seong, Chi-Nam;Han, Ji-Man;Yoo, Jin-Cheol
    • Journal of Microbiology and Biotechnology
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    • 제14권3호
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    • pp.576-583
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    • 2004
  • GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules and shows antimicrobial activities against Gram-positive bacteria. Deoxysugar biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-l55 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP g]ucose-4,6-dehydratase gene was designated as gerE. GerE was expressed in E. coli by using a recombinant expression vector pHJ1. The expressed protein was purified from E. coli cell lysate by using ammonium sulfate fractionation, and DEAE-sepharose CL-6B and hydroxylapatite column chromatography. The molecular mass of the expressed protein correlated with the predicted mass that was deduced from the cloned gene sequence data. The recombinant protein was a homodimer with a subunit relative molecular weight of 39,000 Dalton. It was found to have dTDP-glucose 4,6-dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of $K_{m} and V_{max}$ for dTDP-g]ucose were $32\mu$M and 335 nmol $min^{-1}$ (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of the protein. $NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

ATF2 전사인자의 발현과 AP-1 전사인자인 BATF, c-Fos, c-Jun과의 이량체 형성 (Expression of ATE2 Transcription Factor and the Interaction with AP-1 Factors : BATF, c-Fos, c-Jun)

  • 장혜영;김재호
    • 생명과학회지
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    • 제15권6호
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    • pp.928-934
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    • 2005
  • ATF2는 c-Fos와 c-Jun과 같은 전사인자이며 이들은 루신지퍼 단백질이다. 루신지퍼 단백질은 동형이합체 혹은 이형이합체를 형성하며 promoter 영역에 결합하여 전사조절에 중요한 역할을 한다. 세포의 전사인자 ATF2는 ATF/CRE site에 결합하며 특히 선택적인 interfamily 이형이량체를 형성함으로써 전사 조절에 있어서 다양한 메카니즘을 제공할 수 있다. 본 연구에서는 ATF2 cDNA를 6xHis를 가진 expression vector에 subcloning하여 E.cnli BL2l에서 발현시켰다. 6xHis tag은 nickel-chelating chromatography를 가능하게 하였다 발현된 ATF2는 In vitro binding pull-down assay에서 동형이합체를 이를 뿐만 아니라 AP-1 그룹의 인자들과 선택적인 이형이합체를 형성함을 보여 주었다. BATF와는 강하게 결합하였으며 c-Jun과도 안정된 이합체를 형성하였다. 그러나 c-Fos와는 이합체를 형성하지 않으므로서 AP-1그룹 내에서도 이합체 형성이 선택적으로 이루어짐을 알 수 있다.

Structural Studies on the E. coli Methionyl-tRNA Synthetase and Their Interaction with E. coli $tRNA^{fMet}$

  • Kim Ji-Hun;Ahn Hee-Chul;Park Sung-Jin;Kim Sung-Hoon;Lee Bong-Jin
    • 한국자기공명학회논문지
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    • 제9권2호
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    • pp.110-121
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    • 2005
  • E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.

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Partial Purification and Characterization of Superoxide Dismutase from Tomato (Lycopersicon esculentum) Fruit

  • Kumar, Sunil;Dhillon, Santosh;Singh, Dharam;Singh, Randhir
    • Preventive Nutrition and Food Science
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    • 제9권3호
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    • pp.283-288
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    • 2004
  • Superoxide dismutase (SOD) from tomato (Lycopersicon esculentum Mill.) fruit was purified by ammonium sulphate precipitation, Sephadex G-100 and DEAE-cellulose column chromatographies. A 22 fold purification and an overall yield of 44% were achieved. The purified enzyme was a homodimer with Mr 37.1 kDa and subunit Mr 18.2 kDa as judged by SDS-PAGE. SOD showed $K_{m}$ values of 25 ${\times}$ 10$^{-6}$ M and 1.7 ${\times}$ 10$^{-6}$ M for nitroblue tetrazolium (NBT) and riboflavin as substrates, respectively. The enzyme was thermostable upto 5$0^{\circ}C$ and exhibited pH optima of 7.8. The effect of metal ions and some other compounds on enzyme activity was studied. $Co^{2+}$ and $Mg^{2+}$ were found to enhance relative enzyme activities by 27 % and 73 %, respectively, while M $n^{2+}$ inhibited the SOD activity by 64%. However, $Ca^{2+}$ and C $u^{2+}$ had no effect on enzyme activity. Other compounds like $H_2O$$_2$ and Na $N_3$ inhibited enzymatic activities by 60% and 32%, respectively, while sodium dodecyl sulphate (SDS), chloroform plus ethanol and $\beta$-mercaptoethanol had no effect on the activity of SOD. of SOD.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1

  • Kim, Subin;Sung, Jongmin;Yeon, Jungyoon;Choi, Seung Hun;Jin, Mi Sun
    • Molecules and Cells
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    • 제42권6호
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    • pp.460-469
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    • 2019
  • Bacterial ${\alpha}-type$ carbonic anhydrase (${\alpha}-CA$) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable ${\alpha}-CA$ from Persephonella marina EX-H1 (pmCA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pmCA homodimer in which each monomer consists of a 10-stranded ${\beta}-sheet$ in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pmCA and solvent molecules. These findings may assist development of novel ${\alpha}-CAs$ with improved thermal and/or alkaline stability for applications such as $CO_2$ capture and sequestration.

Mannan-binding lectin of the sea cucumbers Stichopus japonicus has common antigenic determinants with human serum mannan-binding lectin

  • Bulgakov, A.A.;Petrova, I.Yu.;Vakhrusheva, N.M.;Eliseikina, M.G.
    • 한국어업기술학회:학술대회논문집
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    • 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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    • pp.530-530
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    • 2000
  • The host defense system or immune system of all modern animals has their roots in very ancient organisms. After analyzing literature data concerning properties of invertebrates and vertebrates lectins we suggest that mechanism of mannans recognition may exist in marine invertebrates, as a universal mechanism for homeostasis maintenance and host defense, and mannan-binding lectins family of vertebrates has ancient precursor, as was shown for another S-type lectins family. We carried out the screening of mannan-binding type lectin among different species of echinoderms inhabiting in Piter the Grate Bay, the sea of Japan. As a result, the C-type lectins (SJL-32) specific for high mannose glycans was isolated from the coelomic plasma of the sea cucumbers Stichopus japonicus by ion-exchange chromatography on a DEAE-Toyopearl 650M, affinity chromatography on a mannan-Sepharose 6B and gel filtration on a Sephacryl S-200. SJL-32 is homodimer with molecular mass about 32 kDa on SDS-PAGE under non-reducing conditions. Protein part of the lectin has high conteins Asn, Glu, Ser. Hemagglutination of trypsin-treated O blood group human erythrocytes by SJL-32 was competitively inhibited by high-branched -D-mannan composed of -1,2 and -1,6 linked D-mannopyranose residues. In contrast, a variety of mono-, oligo-, and polysaccharides composed of residues of galactose and fucose showed absence or little inhibitory activities. The lectin activity strong depends on Ca2+ concentration, temperature and pH. Monospecific polyclonal antibodies were obtained to the lectin. As was shown by ELISA assay, antibodies to SJL-32 cross-reacted with human serum mannan-binding lectin. This data allows making conclusion about common antigenic determinants and structural homology of both lectins. In our opinion, SJL-32 belongs to evolutionary high conservative mannan-binding lectins (MBLs) family and takes part in the host defense against pathogenic microorganisms.

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