• 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.

• 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.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.372-378
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• 1996

The effects of branched chain amino acids and metabolites in growth media on the biosynthesis of Serratia marcescens threonine dehydratase activity were examined. The enzyme activity was decreased above 60% by leucine among the range from 1 to 20 mM, and the enzyme activity was decreased approximately 20% by a low concentration of valine (1 to 4 mM), but not affected at high concentration (20 mM). However, the enzyme activity was increased approximately 100 to 140% by a low concentration of isoleucine (1 to 4 mM), but decreased approximately 25 to 80% at high concentration (15 to 30 mM). The enzyme activity was decreased by 25 and 58% by the simultaneous addition of all three branched chain amino acids at 2 and 10 mM concentration, respectively, but increased by 75 and 50% by the combination addition of isoleucine plus valine and isoleucine plus leucine at 2 mM, respectively. cAMP was decreased the enzyme activity approximately 10 to 40% by a low concentration (1 to 2 mM), but increased by 80% at high concentration (10 mM). These data suggest that S. marcescens threonine dehydratase should be multivalently repressed by branched chain amino acids, but positively regulated by a low isoleucine concentration and may play a regulatory role in an isoleucine biosynthetic pathway unlike the E. coli K-12 enzyme.

• 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.

• Proceedings of the Korean Journal of Food and Nutrition Conference
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• 2001.12a
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• pp.121-121
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• 2001

최소 배지에 여러 아미노산과 대사 산물을 첨가하여 배양시킨 Serratia marcescens ATCC 25419 세포추출물에서여 biodegradative threonine dehydratase (BDTD), biosynthetic threonine dehydratase (BSTD)와 acetolactate syntase (ALS)의 비활성도를 조사하였다. S. marcescens BDTD와 ALS는 낮은 농도 (0.5-2 mM)의 cAMP에 의해 촉진적 조절을 받으며, 비교적 낮은 농도의 isoleucine (1-4 mM)에 의해서는 S. marcescens BSTD의 생합성이 증가되고 높은 농도의 isoleucine (10-30 mM)에서는 감소되고 비교적 낮은 농도의 valine (2-4 mM)에 의해서 S. marcescens ALS의 생합성이 증가되는 것으로 보아 S. marcescens ATCC 25419에서 branched chain 아미노산 생합성 과정의 조절 양상은 Escherichia coli K-12와는 달리, isoleucine의 생합성 과정은 BSTD에 의해 조절되고, valine의 생합성 과정은 ALS에 의해 조절되는 것으로 사료된다.

• 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.

• BMB Reports
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• v.28 no.2
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• pp.124-128
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• 1995

Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.10 no.7
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• pp.1766-1772
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• 2009

In this study, we are interested in comparing the protein profiles of acid-shocked and control cells of S. mutans isolated from Korean children with caries. The results of 2D gel electrophoresis showed that twelve proteins are up-regulated when the cells were grown under 20 mM lactic acid stress in the exponential phase. Up-proteins under acid stress were estimated a major key of the survival and proliferation of S. mutans in low pH environments. These proteins are estimated generally associated with three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis.