• The Korean Journal of Food And Nutrition
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• v.23 no.2
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• pp.171-177
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• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.660-664
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• 2000

To clone genes related UK-58,852 production, genomic DNA of strain Actinodura roseorufa was used for the construction of genomic library using pOJ446 cosmid vector. The genomic library was screened rising dehydratase PCR product and eryA gene as a DNA hybridization probe. pHD54 was isolated, which contained an approximately 35kb of inserted DNA. BamHI, SmaI and sonicater fragments hybridized to eryA probe. All of pHD54 BgmHI, SmaI and sonicater fragments were subcloned into pGEM7 and some fragments which hybridized to eryA probe were sequenced. The nucleotide sequence was analysed using BLAST program. The sequence identities were observed in KS,AT, KR, ER and PKS loading domains. Also oxidoreductase showed similarity to rifamycin module10, and dTDP-D-glucose 4,6 dehydratase and TDP-D-glucose synthase involved in biosynthesis of sugar showed similarity to Streptomyces argillaceus.

• BMB Reports
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• v.28 no.2
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• pp.118-123
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• 1995

Biodegradative threonine dehydratase was purified to homogeneity from Serratia marcescens ATCC 25419 by streptomycin sulfate treatment, Sephadex G-200 gel filtration chromatography followed by AMP-Sepharose 4B affinity chromatography. The molecular weight of the purified enzyme was 118,000 by fast protein liquid chromatography using superose 6-HR. The enzyme was determined to be a homotetrameric protein with subunit molecular weights of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by ${\alpha}-Keto$ acids and their derivatives such as ${\alpha}-ketobutyrate$, pyruvate, glyoxlyate, and phosphoenol pyruvate, but not by ${\alpha}-aminobutyrate$ and ${\alpha}-hydroxybutyrate$. The inhibition of the enzyme by pyruvate and glyoxylate was observed in the presence of AMP. The inhibitory effect of glyoxylate was decreased at high enzyme concentration, whereas the inhibition by pyruvate was independent of the enzyme concentration. The kinetics of inhibition of the enzyme by pyruvate and glyoxylate revealed a noncompetitive and mixed-type inhibition by the two inhibitors with respect to L-threonine and AMP, respectively.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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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.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.749-754
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• 2000

PCR primers were designed based on consensus sequences of dTDP-D-glucose 4,6-dehydratase, one of the enzymes involved in the biosynthesis of deoxysugar. The PCR product (360 bp) was obtained from Thermus caldophilus GK24. Colony hybridization was carried out to the cosmid library constructed from T. caldophilus GK24 genomic DNA by the PCR product DNA fragment. We isolated a cosmid clone (pSMTC-1) that was subcloned to call pKCB series plasmid (BamHI fragments), partially sequenced and analyzed. pKCB80 (4.2 kb-BamHI DNA fragment) of them showed ORFs that was orfA, orfB, orfC and orfD. The orfABCD gene cluster is the deosysugar biosynthetic gene ; orfA (glucose-1-phosphate thymidylytransferase), orfB (dTDP-D-glucose 4,6-dehydratase), orfC (dTDP-4-keto-L-rhamnose reductase) and orfD (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase). The gene cluster that was related in biosynthesis of dTDP-L-rhamnose was also identified by computer analysis, and we proposed that the biosynthetic pathway of deoxysugar analyzed from DNA sequencing of pKCB80 is from D-glucose-1-phosphate, dTDP-D-glucose, dTDP-4-keto-6-deoxy-D-glucose via dTDP-4-keto-L-rhamnose to dTDP-L-rhamnose.

• BMB Reports
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• v.31 no.5
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• pp.475-483
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• 1998

Many antibiotics contain partially deoxygenated sugar components that are usually essential for biological activity, affinity, structural stability, and solubility of antibiotics. Gene probes of the biosynthetic genes related with the deoxysugar were obtained from PCR. Primers were designed from the conserved peptide sequences of the known dTDP-D-glucose 4,6-dehydratases, which are the key step enzymes in the biosynthesis of deoxysugar. The primers were applied to amplify parts of dehydratase genes to 27 actinomycetes that produce the metabolites containing deoxysugar as structural constituents. About 180 and 340 bp DNA fragments from all of the actinomycetes were produced by PCR and analyzed by Southern blot and DNA sequencing. The PCR products were used as gene probes to clone the biosynthetic gene clusters for the antibiotic mithramycin, rubradirin, spectinomycin, and elaiophyrin. This method should allow for detecting of the biosynthetic gene clusters of a vast array of secondary metabolites isolated from actinomycetes because of the widespread existence of deoxysugar constituents in secondary metabolites.

• Proceedings of the Zoological Society Korea Conference
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• 1998.10b
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• pp.268.1-268
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• 1998

No Abstract, See Full Text

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.174-179
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• 1990

In order to overproduce L-phenylalanine, various kind of regulatory mutants were isolated from parental Escherichia coli K-12. MWEC 83 Producing 7.4g/l of L-phenylalanine has been derived as a tyrosine and tryptophan double auxotrophic mutant. To produce L-phenylalanine without adding L-tyrosine and L-tryptophan, revertant strain MWEC 101 was isolated from MWEC 83. Further various analogues and valine resistant mutants were isolated from MWEC 101. MWEC 101-5 was the most excellent strain that produced 17.9g/l of L-phenylalanine after having been cultivated for 54 hours in 15% glucose medium. It was disclosed that activities of rate-limiting enzymes including chorismate mutase and prephenate dehydratase in MWEC 101-5 were desensitized to 2mM L-phenylalanine in the enzyme reaction mixture and that activities level of 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase and prephenate dehydratase were increased more than 20 times over those of the parental strain.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1595-1605
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• 2023

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQD^WT with the citrate at a resolution of 1.80Å and CgDHQD^R19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQD^S103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQD^S103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

• Journal of Nutrition and Health
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• v.24 no.6
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• pp.526-533
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• 1991

The effect of dietary selenium on the activity of $\delta$-aminolevulinic acid dehydratase (ALAD) inhibited by the administration of lead were investigated in rats. The levels of dietary lead in the acetate form were 0(contro)200, 1, 000, 2, 000 and 5, 000ppm. Except control group four-level of lead diet groups were again subdivided into two depending on with and without 0.5ppm selenium supplementation. Sixty-three 40-day-old male Sprague-Dawley rats weighing 141$\pm$5g were distributed into total of nine diet groups according to RCB design and fed ad libidum for 4 weeks. Lead dietary groups did not show any significnat difference in food intake from the control group. Food efficiency and weight gain were lower in 2.000ppm and 5, 000ppm lead groups but not found in selenium supplemented ones. Hemoglobin contents hematocrit values ALAD activities in blood were significantly decreased and urinary aminolevulinic acid(ALA) excretion increa-sed with increasing dietary lead levels but partly restored by selenium supplementation. however only in 200, 1, 000 and 2, 000ppm dietary lead groups. On the other hand the hepatic ALAD activites of all four lead groups were recovered 19-30% from suppression by selenium supplementation. It was concluded that selenium administration alleviated lead toxicity in rats.