• KOREAN JOURNAL OF CROP SCIENCE
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• v.40 no.1
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• pp.52-58
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• 1995

5-methyltryptophan（5MT） resistant mutant plants （MRl） were analyzed for characterization of anthranilate synthetase （AS） and tryptophan synthetase （TS） enzymes. The enzyme was measured in crude extracts from MR1 and control seedlings of Danggin inbred line. There was no significant difference in the level of AS between MR1 and control seedlings when grown on MS medium without 5MT. However, MR1 seedlings grown on MS medium with 25mg/L 5MT showed the level of AS twice higher than that of control seedlings. The activity of AS was inhibited to 50％ in untreated plants when 4mg /L L-tryptophan was added to their extracts. Extracts from MR1 plants required about four times higher concentration of amino acid to cause equal inhibition. In the TS assay, the activity observed in MR1 seedlings was four times higher than that of control seedlings. We have also isolated and sequenced the gene which encoding the tryptophan synthetase B subunit （TSB） from maize. The gene encodes polypeptides with high homology to TSB isolated from other plants, and is expressed in all the developmental stages examined. Northern hybridization analysis indicated that the gene expression in MR1 seedlings grown on MS medium showed a higher level than in control seedlings.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.798-803
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• 2001

Peptide synthetases produced from various microorganisms are multifunctional enzyme complexes and their substrates are recognized and activated by adenylation domains. To identify the substrate specificity of the peptide synthetase isolated from Bacillus subtilis 713, known to produce an antifungal peptide, two adenylation domains containing the minimal functional portion were expressed and purified. ATP-ppi exchange experiments and kinetic studies revealed that the two adenylation enzymes had a substrate specificity to $_{L}-lysine{\;}and{\;}_{L}-tryptophan$, respectively. In addition, based on a signature sequence comparison, the substrate of the third domain was predicted to be L-glutamic acid. These results suggest that this peptide synthetase is novel because there has been no previous report on a peptide synthetase that uses $_{L}-lysine,{\;}_{L}-tryptophan,{\;}and{\;}_{L}-glutamic$ acid as substrates in that order.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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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.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.564-569
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• 1992

A green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum NCIB 8327, was grown in modified Pfennig's medium including glu1amate as a nitrogen source. Glutamine synthetase was isolated through a series of ultracentrifugation. DEAE-Sepharose CL-6B ion exchange chromatography. Sephacryl S-300 gel permeation chromatography, and preparative HPLC. The recovery and purification fold of the enzyme were 2% and 46.3. respectively. The isolated enzyme was homogeneous on UV-Visible spectrum and polyacrylamide gel electrophoretogram. The relative molecular mass of the native enzyme was estimated to be 280,000 by gel permeation chromatography. The enzyme consisted of ten subunits with relative similar molecular mass. 30.000. which was estimated by SDS-polyacrylamide gel electrophoresis. The optimal temperature and pH of the enzyme were $30^{\circ}C$ and 7.0. Km values were 27.9 mM for L-glutamine and 0.92 mM for hydroxylamine-HCr. The enzyme activity was inhibited by alanine. glycine. and tryptophan considerably, but was not affected by asparagine, lysine. leucine. and valine.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.427-432
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• 1986

The regulation of three ammonia assimilatory enzymes, GDH (glutamate dehydrogenase), GS (glutamine synthetase) and GOGAT (glutamate synthase), has been examined in C. glutamicum. Three kinds of arginine auxotrophs blocked in each step of arginine biosynthetic pathway from glutamate were selected as arg 5, arg 6, arg 8. Histidine and tryptophan auxotrophs were also selected because histidine and tryptophan repressed GS biosynthesis in E. coli. These strains were cultured on the media containing nitrogen-excess and limited conditions, to compare the specific activities of ${\alpha}$-ketoglutarate dehydrogenase(${\alpha}-KGDH$), GDH, GS, GOGAT from the cell-free extracts. These results showed that enzyme levels of ${\alpha}-KGDH$ and GDH from 3 kinds of arginine auxotrophs, histidine and tryptophan auxotrophs in nitrogen-excess condition and those of GS and GOGAT in nitrogen limited condition were increased compared with opposite condition. The tryptophan and histidine auxotrophs showed higher level of glutamate and glutamine than parental strains and other mutants. it is assumed that the higher levels of ${\alpha-KGDH}$ and GDH from mutants in nitrogen-excess condition promoted the accumulation of glutamate and glutamine in fermentation broth. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with histidine, tryptophan, glycine, alanine, serine and GMP implied that a system of feedback inhibition were effective. The GDH, GS and GOGAT biosynthesis in culture broth was markedly repressed by the nature and kinds of available nitrogen sources such as tryptophan, proline, glycine, alanine, serine and tyrosine.

• BMB Reports
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• v.29 no.4
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• pp.277-285
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• 1996

In Pseudomonas fluorescens grown on malonate as sole carbon source, acetyl-CoA synthetase was induced, suggesting that malonate is metabolized through acetate and then acetyl-CoA. Acetyl-CoA synthetase was purified 18.6-fold in 4 steps to apparent homogeneity. The native molecular mass of the enzyme estimated by a native acrylamide gel electrophoresis was 130 kDa. The enzyme was composed of two identical subunits with a molecular mass of 67 kDa. Optimum pH was 70. The acetyl-CoA synthetase showed typical Michaelis-Menten kinetics for the substrates, acetate, ATP and CoA, whose $K_m$ values were calculated to be 33.4, 74.8, and 40.7 mM respectively. Propionate. butyrate and pentanoate were also used as substrates by the enzyme, but the rate of the formation of the CoA derivatives was decreased in the order of the increase in carbon number. The enzyme was inhibited by the group-specific reagents diethylpyro-carbonate, 2,3-butanedione, pyridoxal-5'-phosphate and N-bromosuccinimide. In the presence of substrates the inactivation rate of the enzyme, by all of the group-specific reagents mentioned above decreased, indicating the presence of catalytically essential histidine, arginine, lysine and tryptophan residues at or near the active site. Preincubation of the enzyme with ATP, $Mg^{2+}$ resulted in the increase of its susceptibility to diethylpyrocarbonate, suggesting that ATP, $Mg^{2+}$ may induce a conformational change in the active site exposing the essential histidine residue to diethylpyrocarbonate. The enzyme was acetylated in the presence of acetyl-CoA, indicating that this is one of acyl-enzyme.

• Biomedical Science Letters
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• v.13 no.2
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• pp.105-110
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• 2007

Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition. The discriminating enzyme (D-AspRS) recognizes only tRNA$^{Asp}$, while the non-discriminating one (ND-AspRS) also recognizes tRNA$^{Asn}$ and therefore forms both Asp-tRNA$^{Asn}$ and Asp-tRNA$^{Asp}$. Plus primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial-type, larger AspRS (about 580 aa) in addition to a shorter archaeal/eukaryotic type (about 430 aa). In vivo data made clear that discriminating and non-discriminating enzymes exist in both groups. The determinants in the protein sequence responsible for tRNA discrimination are not hewn. The AspRS from Acidithiobacillus ferrooxidans might be suggested ND-AspRS fur missing of AsnRS in genomic sequencing data. Therefore, we analyzed the AspRS from A. ferrooxidans with in vitro aminoacylation assay with E. coli unfractionated tRNA, in vivo missense suppression assay with tipA34 mutant and Northern hybridization with probes which were specific with tRNA$^{Asp}$ or tRNA$^{Asn}$. The AspRS from A. ferrooxidans produced more Asp-tRNA than that from E. coli. Only aspS gene from A. ferrooxidans suppressed trpA34 strain in minimal media without tryptophan. Only AspRS from A. ferrooxidans showed mischarged Asp-tRNA$^{Asn}$ band. Therefore, AspRS from A. ferrooxidans is definitely ND-AspRS.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.89-93
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• 1990

To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.