• Journal of Microbiology
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• v.43 no.3
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• pp.277-284
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• 2005

The avermectins are composed of eight compounds, which exhibit structural differences at three positions. A family of four closely-related major components, A1a, A2a, B1a and B2a, has been identified. Of these components, B1a exhibits the most potent antihelminthic activity. The coexistence of the '1' components and '2' components has been accounted for by the defective dehydratase of aveAI module 2, which appears to be responsible for C22-23 dehydration. Therefore, we have attempted to replace the dehydratase of aveAI module 2 with the functional dehydratase from the erythromycin eryAII module 4, via homologous recombination. Erythromycin polyketide synthetase should contain the sole dehydratase domain, thus generating a saturated chain at the C6-7 of erythromycin. We constructed replacement plasmids with PCR products, by using primers which had been derived from the sequences of avermectin aveAI and the erythromycin eryAII biosynthetic gene cluster. If the original dehydratase of Streptomyces avermitilis were exchanged with the corresponding erythromycin gene located on the replacement plasmid, it would be expected to result in the formation of precursors which contain alkene at C22-23, formed by the dehydratase of erythromycin module 4, and further processed by avermectin polyketide synthase. Consequently, the resulting recombinant strain JW3105, which harbors the dehydratase gene derived from erythromycin, was shown to produce only C22,23-unsaturated avermectin compounds. Our research indicates that the desired compound may be produced via polyketide gene replacement.

• Korean Journal of Crystallography
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• v.7 no.2
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• pp.120-125
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• 1996

Single crystals of deoxy-thymidine diphosphate-D-gluxose-4,6-dehydratase(abbreviated as dTDP-D-glucose dehydratase) from Escherichia coli Strain BL21 clone which harbors the gene of dTDP-D-glucose dehydratase in Salmonella typhimurium LT2 have been grown with and whithout substrates by sitting drop vapor diffusion at room temperature. The precipitating agent was 1.6 to 2.0 M Na, K phosphate buffer(pH 8.0). The crystals diffract to at least 2.5Å and belong to the hexagonal space group P61 with cell dimensions a=b=168.54Å, c=81.08Å. The asymmetric unit contains one dimer with a crystal volume per protein mass(VM) of 2.4Å3/Da and solvent content (Vsol) of 64% by volume.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.238-245
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• 1988

Studies were made on the regulation of chorismate mutase and prephenate dehydratase of a species of Intrasporangium, a phenylalanine producing Actinomycete isolated from soil. Two distinctly regulated species of chorismate mutase, designated CM I and CM IIwere resolved by DEAE Cellulose and DEAE Sephadex A 50 chromatography. The activity of CM II was inhibited by L-tyrosine, whereas that of CM I appeared to be unregulated. Single species of prephenate dehydyatase was also separated in the same purification steps. The activity of the enzyme was strongly feedback inhibited by L-phenylalanine, but by L-tyrosine or L-methionine it was rather slightly stimulated. Synthesis of chorismate mutase was not influenced by the presence of phenylalanine, tyrosine or tryptophan, whereas prephenate dehydratase was found to be subject to strong feedback repression by L-phenylalanine. The rate of repression was 94% at the concentration of 1mM L-phenylalanine but the repression was completely offset by the presence of 5mM tyrosine. The critical regulatory site of the phenylalanine terminal biopathway was, therefore, proved to be the second reaction which was catalyzed by the L-phenylalanine inhibitable and repressible prephenate dehydratase.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.24-30
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• 1995

The effects of amino acids in growth media on the biosynthesis of Serratia marcescens biodegradative threonine dehydratase activity were examined. The enzyme activity was decreased by 44 and 34% by 10 mM isoleucine and valine, respectively, whereas it was increased approximately by 20% by 10 mM threonine. Among several metabolites tested, pyruvate increased the enzyme activity by 60% at 5 mM, but decreased the enzyme activity approximately by 20 to 70% above 20 mM. The enzyme activity was increased by 64% by 5 mM glyoxylate, whereas it decreased the enzyme activity approximately by 40 to 70% above 20 mM glyoxylate. The thiamine, monopyrrole derivative, also increased the enzyme activity by 84% at 50 $\mu $g/ml, but did not affected the enzyme activity above 300 $\mu $g/ml. cAMP increased the enzyme activity by 58% at 0.5 mM, but decreased the enzyme activity by 15% at 2 mM. These data suggested that the biosynthesis of Serratia marcescens biodegradative threonine dehydratase is regulated by concentrations of pyruvate, glyoxylate and cAMP.

• Journal of Microbiology and Biotechnology
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• v.5 no.5
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• pp.305-308
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• 1995

From the plasmid library made from Sstl and San-digested genomic DNA of Streptomyces fradiae NRRL 2702, four positive clones were selected using an oligodeoxynucleotide probe from the N-terminal amino acid sequence of purified threonine dehydratase. The cloned gene for threonine dehydratase was a 2.0 kilo-base pair DNA fragment. The deduced amino acid sequence of PCR product (PCR245) was matched to that of the N-terminal part of threonine dehydratase from S. fradiae and this showed a high similarity to the threonine dehydratases of other organisms. This indicated that amino acid sequences of threonine dehydratases were highly conserved and the polypeptide product of the PCR245 was likely to be involved in the deamination of threonine.

• BMB Reports
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• v.32 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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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.

• YAKHAK HOEJI
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• v.19 no.1
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• pp.21-29
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• 1975

The activity of $\delta$-aminolevlinic acid dehydratase (ALAD) in red cell of rabbit inhibited by addition of $Pb(Ac)_{2}$(50mg/kg) to rabbit caused to diminish completely the ALAD activity in blood within shr. Pretreatment of ascorbic acid and methionine decreased the increment of $\delta$-aminolevulinic acid output in urine by lead poisoning.

• BMB Reports
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• v.29 no.3
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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.