• Journal of Life Science
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• v.11 no.1
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• pp.43-47
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• 2001

A mutant tryptophan synthase $\alpha$-subunit, where Pro28 was replaced with Leu, tends to be expressed in recombinant E. coli. CD and fluorescence spectra of this protein indicate some changes in secondary and tertiary structure. Wild type protein was more or less affected by {TEX}$Ca^{2+}${/TEX} ion in regards of the fluorescent properties of its native, unfolded and intermediate forms, but the mutant protein was not at all. The dramatic structural changes may be related to the aggregation of this mutant protein.

• Journal of Life Science
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• v.14 no.1
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• pp.14-16
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• 2004

Various ligands function as regulators in the allosteric control of tryptophan synthase. Effects of the monovalent cations and glycerophosphate on the mutant tryptophan synthases were examined in the presence of L-serine. The results showed that these compounds might play roles in the allosteric control of the proteins.

• Journal of Life Science
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• v.12 no.4
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• pp.464-468
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• 2002

Enzymatic activities and fluorescence spectroscopic properties of the double mutant proteins P96L/F139W, P96L/F258W and a triple mutant protein P96L/F139W/F258W of tryptophan synthase $\alpha$ subunit from Escherichia coli was examined to study tertiary and local structure changes around the tryptophan residues. The enzymatic activities of P96l./F139W and P96L/F258W were similar, but P96L/F139W/F258W had lower activity, as compared to wild type. The fluorescence intensities of double mutant, P96L/F139W and P96L/F258W, were decreased but that of a triple mutant, P96L/F139W/F258W, was increased when compared to wild type. The sum of the maximum fluorescence intensity (fluorescence intensity at the λ$_{max}$) for the double mutant proteins was not equal to the intensity seen in the triple mutant protein. The enzymatic activity and fluorescence data indicate that the replacement of Pro$^{96}$ longrightarrowLeu might affect on the stability of helix 8 and the loop located between strand 4 and helix4. The result suggests that the tertiary structure of triple mutant (P96L/F139W/F258W), being different from wild type, might have more compact residual structure at the vicinity of 139 and 258.8.

• Journal of Plant Biology
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• v.39 no.4
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.

• BMB Reports
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• v.28 no.1
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• pp.40-45
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• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• Journal of Life Science
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• v.6 no.4
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• pp.304-312
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• 1996

In the present report, a p[ossibility of the interaction fo Ser-33 and Asp-112 residues in folding of tryptophan synthase $\alpha$ subunit was explored by examining the effect of single or double substitution of these residues on folding of $\alpha$ subunit in E. coli. $\alpha$ subunit of which Ser-33 was substituted with Leu (SL33) was accumulated as insoluble aggregate form, when overproduced in E. coli, whereas $\alpha$ subunit of which Asp-112 was replaced by Asn (DN112) or Gly (DG112) was accumulated as soluble form to the similar extent as wild type $\alpha$ subunit was. When these alterations were combined into one protein, the synergistic effect of residues 33 and 112 on the amount of aggregate form was shown. The amount of doubly altered SL33/DG112 $\alpha$ subunit as aggregate form was increased 5-13 fold that of SL33 $\alpha$ subunit, and the amount of SL33/DG112 $\alpha$ subunit as aggregate form was decreased 3-4 fold that of SL33 $\alpha$ subunit. Aggregates are derived from the specific association of partially folded or unassembled subunits in the folding process. Therefore, this result suggests that residues 33 and 112 of $\alpha$ subunit may unteract during the folding of this enzyme in E. coli.

• Journal of Life Science
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• v.14 no.1
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• pp.17-20
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• 2004

Effects of monovalent cations were examined on the fast $\beta$reaction of $\alpha$D56N and $\alpha$D56G mutant tryptophan synthase. Reaction rates for the production and degradation of intermediates in the reaction were changed in the presence of cathons. The mutant proteins showed different reaction rates from those of wild-type protein, and additional changes occurred in the presence of cations. The results showed that monovalent cations and $\alpha$D56 are important in allosteric properties of this protein.

• 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.

• The Korean Journal of Zoology
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• v.24 no.3
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• pp.123-131
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• 1981

The purple $(pr^+)$ gene of Drosophila and its associated enzyme, sepiapterin synthase, were employed in a study of the relationship between ontogenic expression and the location of the gene in the genome, Enzyme assays performed at different developmental stages indicate the $T(Y:2)pr^c5, cn/pr^c4 cn$ flies (files in which $pr^+$ has been translocated and which exhibit variegation) have a reduced amount of enzyme activity as compared with both wild-type and $pr^1$ flies. This reduction in activity was not found in larval stages, which suggests that the inactivation process probably occurs in late larval or early pupal stages. Tryptophan pyrrolase, the enzyme system associated with vermilion $(v^+)$, was also examined for activity in different developmental stages of the fly. Genotype carrying a translocated $v^+$ shows a peak of tryptophan pyrrolase activity in late larval stages, whereas, Oregon-R exhibits the lowest activity at this period.