• BMB Reports
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• v.32 no.1
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• pp.20-24
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• 1999

The first step of the branch pathway in tryptophan biosynthesis is catalyzed by anthranilate synthase, which is subjected to feedback inhibition by the end product of the pathway. The $trpE^{FBR}$ gene from a mutant Escherichia coli strain coding for anthranilate synthase that was insensitive to feedback inhibition by tryptophan has been cloned. To identify the amino acid changes involved in the feedback regulation of anthranilate synthase, the nucleotide sequence of the mutant $trpE^{FBR}$ gene was determined. Sequence analysis of the $trpE^{FBR}$ gene revealed that four bases were changed in the structural gene while alteration was not found in the 5' control region. Among these base changes, only two base substitutions caused the alterations in amino acid sequences. From the results of restriction fragment exchange mapping, the 61st nucleotide, C to A substitution, that changed $Pro^{21}{\rightarrow}Ser$ was identified as the cause of the desensitization to feedback inhibition by tryptophan. Additional feedback-resistant enzymes of the E. coli anthranilate synthases were constructed by site-directed mutagenesis to examine the effect of the $Ser^{40}\;{\rightarrow}\;Arg^{40}$ change found in the $trpE^{FBR}$ gene of Brevibacterium lactofermentum. From the feedback inhibition analysis, the $Pro^{21}{\rightarrow}Ser$ and $Ser^{40}{\rightarrow}Arg$ mutants maintained about 50% and 90% of their maximal activities, respectively, even at the extreme concentration of 10 mM tryptophan. From these results, we suggest that the $Pro^{21}$ and $Ser^{40}$ residues are involved in the tryptophan binding in the E. coli enzyme.

• 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.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.186-195
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• 2015

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive ${\alpha}$ subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

• 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.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.10a
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• pp.127-127
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• 2003