ArticlePlant physiology2005
Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.
Article in Plant physiology, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 29 citations in OpenAlex.
- Genome Editing Technology and Its Application to Metabolic Engineering in Rice.Rice (New York, N.Y.) · 2022Review
- Catalytic promiscuity of rice 2-oxoglutarate/Fe(II)-dependent dioxygenases supports xenobiotic metabolism.Plant physiology · 2021Article
- TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.Fungal genetics and biology : FG & B · 2016Article
- Changes in primary and secondary metabolite levels in response to gene targeting-mediated site-directed mutagenesis of the anthranilate synthase gene in rice.Metabolites · 2012Article
- Application of gene targeting to designed mutation breeding of high-tryptophan rice.Plant physiology · 2011Article
- Article
- Practical cell-free protein synthesis system using purified wheat embryos.Nature protocols · 2010Article
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
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