• 제목/요약/키워드: dTDP-glucose

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Cloning and Idendification of dTDP-L-Rhamnose Biosynthetic Gene Cluster from Thermus caldophilus GK24

  • Kim, Ki-Chan;Lee, Seung-Don;Han, Ju-Hee;Sohng, Jae-Kyung;Liou, Kwang-Kyoung
    • 한국생물공학회:학술대회논문집
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    • 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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    • pp.749-754
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    • 2000
  • 알려진 dTDP-D-glucose 4,6-dehydratase의 amino acid 서열로 부터 primer를 제작하여 내열성 균주인 Thermus caldophilus GK24에서 colony hybridization 과정을 거쳐 dTDP-D-glucose 4,6-dehydratase를 포함하는 cosmid DNA를 얻었다. 유전자 분석을 위해 cosmid DNA를 subclone 하여 작은 크기로 분리하였다. 분리된 cosmid를 pSMTC-1 으로 명명하고 pSMTC-1를 BamHI으로 반응시켜 BamHI 단편 모두를 pGEM 7(+)를 이용하여 subclone 하였다. 각각의 이름은 크기에 따라 pKCB10(1.2kb-BamHI), pKCB20(1.6kb-BamHI), pKCB30(2.Ikb-BamHI), pKCB40(2.5kb-BamHI), pKCB50(2.5kb-BamHI), pKCB60(2.7kb-BamHI), pKCB70(3.4kb-BamHI), pKCB80(4.4kb-BamHI), pKCB90(7.0kb-BomHI) 으로 명명하였다. 각각의 subclone된 유전자를 분석하기 위해 Erase-a-base 방법을 이용하여 template를 준비하였고 이를 자동 염기서열 분석기를 이용하여 염기서열을 분석하였다. 염기서열분석 결과 pKCB80(4.2kb)에 dTDP-D-glucose synthase(orfA) 유전자를 비롯하여 dTDP-D-glucose 4,6-dehydratase(orfB), orfC (dTDP-4-keto-L-rhamnose reductase) 그리고 orfD(dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase)와 유사한 유전자들이 있음이 확인 되었고 dTDP-L-rhamnose의 생합성 과정을 예상할 수 있었다.

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Function of Lysine-148 in dTDP-D-Glucose 4,6-Dehydratase from Streptomyces antibioticus Tu99

  • Sohng, Jae-Kyung;Noh, Hyung-Rae;Lee, Oh-Hyoung;Kim, Sung-Jun;Han, Ji-Man;Nam, Seung-Kwan;Yoo, Jin-Cheol
    • Journal of Microbiology and Biotechnology
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    • 제12권2호
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    • pp.217-221
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    • 2002
  • dTDP-D-glucose 4,6-dehydratase (TDPDH) catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, and requires $NAD^+$ as a coenzyme for its catalytic activity. The dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ tightly binds $NAD^+$ [19]. In order to determine the role of lysine-148 in the $NAD^+$ binding, the lysine of the dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus $Tu{\ddot}99$ was mutated to various amino acids by site-directed mutagenesis. The catalytic activity of the four mutated enzymes of TDPDH did not recover after addition of $NAD^+$ . However, the activity of K159A, the mutated enzyme of UDP-D-glucose 4-epimerase (UDPE), recovered after the addition of $NAD^+$ [15]. Although dTDP-glucose 4,6-dehydratase, and UDP-galactose (glucose) 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family and the lysine-148 of TDPDH was highly conserved as in UDPE (Lys-159), the function of the lysine-148 of TDPDH was different from that of UDPE. The mutated enzymes showed that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase played no role in the $NAD^+$ binding. Accordingly, it is suggested that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase is involved in the folding of TDPDH.

A New Deoxyhexose Biosynthetic Gene Cluster in Streptomyces griseus ATCC10137: Heterologous Expression of dTDP-D-Glucose 4,6-Dehydratase Gene

  • Kim, Sang Suk;Bang, Jung-Hee;Hyun, Chang-Gu;Kim, Joo-Woo;Han, Jae-Jin;Suh, Joo-Won
    • Journal of Applied Biological Chemistry
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    • 제43권3호
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    • pp.136-140
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    • 2000
  • A novel 6-deoxyhexose biosynthetic gene cluster different from the one for the biosynthesis of streptomycin was isolated from Streptomyces griseus using specifically designed PCR primers to compare the sequence of known dTDP-glucose synthase genes. We cloned a 5.8-kb DNA from Streptomyces griseus ATCC10137, which contained the 4-ketoreductase homologue (grsB), dTDP-glucose synthase (grsD), and dTDP-glucose 4, 6-dehydratase (grsE) genes. Escherichia coli cultures containing plasmid of the PCR product which encoded the grsE region under the controUed T7 promoter were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from TDP-glucose. The enzyme showed high substrate specificity, being specific to only dTDP-glucose that is known to be incorporated into secondary metabolites such as antibiotics.

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Expression of orf7(oxi III) as dTDP-Glucose 4,6-Dehydratase Gene Cloned from Streptomyces antibioticus Tu99 and Biochemical Characteristics of Expressed Protein

  • Yoo, Jin-Cheol;Han, Ji-Man;Sohng, Jae-Kyung
    • Journal of Microbiology and Biotechnology
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    • 제9권2호
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    • pp.206-212
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    • 1999
  • The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

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Mechanism Study of dTDP-D-Glucose 4,6-Dehydratase: General Base in Active Site Domain

  • Sohng, Jae-Kyung;Noh, Hyung-Rae;Yoo, Jin-Cheol
    • BMB Reports
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    • 제32권4호
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    • pp.358-362
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    • 1999
  • dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable sterochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and $NAD^+$ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.

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Biosynthesis of Carbohydrate associated with secondary Metabolites; Biosynthesis of dTDP-4-keto-6-deoxy-D-glucose by four enzyme reaction system

  • 오종민;이선구;김병기
    • 한국생물공학회:학술대회논문집
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    • 한국생물공학회 2001년도 추계학술발표대회
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    • pp.185-188
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    • 2001
  • We have developed a synthetic method for dTDP-4-keto-6-deoxy-D-glucose with four enzyme system. We have used crude extracts from cultures of Escherichia coli BL21 strains harboring plasmids containing different sources. dTDP-4-keto-6-deoxy-D-glucose was synthesized by the combination of thymidine-monophosphate kinase, acetate kinase, dTDP-glucose synthase and dTDP-D-glucose 4,6-dehydratase in a batch system, starting the reaction with dTMP. The enzymatic synthesis strategy allowed a dTMP conversion with a 95%.

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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.

Production of dTDP-4-keto-6-deoxy-D-glucose by Immobilization of dTDP-D-glucose 4,6-dehydratase

  • Kharel, Mandan-Kumar;Liou, Kwang-Kyoung;Sohng, Jae-Kyung;Lee, Hei-Chan
    • Journal of Microbiology and Biotechnology
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    • 제14권2호
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    • pp.297-301
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    • 2004
  • The dTDP-D-glucose 4,6-dehydratase from Salmonella enterica was immobilized using covalent binding to cyanogen bromide activated sepharose. The immobilized enzyme was used to produce dTDP-4-keto-6-deoxy-D-glucose, a key sugar intermediate that can be used economically to produce diverse classes of unusual sugars appended in various antibiotics. The enzyme was immobilized on the sepharose after activation with cyanogen bromide. The maximum immobilization (80.03%) was achieved after 14 h of coupling. The covalently immobilized enzyme was stable, and an average of 78.4 % conversion was achieved until 120 h of immobilization when it was repeatedly used. Similar conversion was noticed for the first batch using the enzyme entrapped-hydrogel but activity was gradually decreased in the following batches. The production of dTDP-4-keto-6-deoxy-D-glucose by using an immobilized enzyme has high potential for commercial application.

Isolation and Characterization of the Biosynthetic Gene Clusters for Aminoglycoside Antibiotics

  • Jung Yong-Gyun;Jo You-Young;Hyun Chang-Gu;Lee In Hyung;Yang Young-Ye1l;Suh Joo-Won
    • 한국미생물학회:학술대회논문집
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    • 한국미생물학회 2001년도 추계학술대회
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    • pp.146-156
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    • 2001
  • The biosynthetic gene clusters for bluensomycin and spectinomycin were isolated and characterized from the bluensomycin producer, Streptomyces bluensis ATCC27420 and the spectinomycin producer, Streptomyces spectabilis ATCC27741, respectively. PCR primers were designed specifically to amplify a segment of dTDP-glucose synthase gene based on its conserved sequences of several actinomycete strains. By screening cosmid libraries using amplified PCR fragments, 30-kb and 45-kb DNA fragments were isolated from Streptomyces bluensis and Streptomyces spectabilis, respectively. Sequencing analysis of them revealed that each contains 15 open reading frames (ORFs). Some of these ORFs were turned out to be antibiotic resistance genes (blmA and speN), dTDP-glucose synthase genes (blmD and spcD), and dTDP-D-glucose 4,6-dehydratase genes (blmE and spcE), suggesting that the blm and spec gene clusters are likely involved in the biosynthesis of bluensomycin and spectinomycin, respectively.

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Cloning, Sequencing and Expression of dTDP-D-Glucose 4,6-Dehydratase Gene from Streptomyces antibioticus $T\ddot{u}99$, a Producer of Chlorothricin

  • Sohng, Jae-Kyung;Yoo, Jin-Cheol
    • BMB Reports
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    • 제29권3호
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    • pp.183-191
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    • 1996
  • DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus $T\ddot{u}99$, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil's function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDP-D-glucose.

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