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Published on July 22, 2005; 10.1104/pp.105.062885

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Received March 15, 2005
Returned for revision May 6, 2005
Accepted May 9, 2005

Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway

Takuya Kanno , Akira Komatsu , Koji Kasai , Joseph G. Dubouzet , Minako Sakurai , Yasuko Ikejiri-Kanno , Kyo Wakasa , and Yuzuru Tozawa *

Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan; Japan Science and Technology Agency for Core Research for Evolutional Science and Technology Plant Functions and Their Control; Mitsubishi Kagaku Institute of Life Sciences, Yokohama Research Center, Yokohama 227-8502, Japan
National Institute of Crop Science, Tsukuba 305-8518, Japan; Japan Science and Technology Agency for Core Research for Evolutional Science and Technology Plant Functions and Their Control
Japan Science and Technology Agency for Core Research for Evolutional Science and Technology Plant Functions and Their Control
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan; Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan; Japan Science and Technology Agency for Core Research for Evolutional Science and Technology Plant Functions and Their Control; Mitsubishi Kagaku Institute of Life Sciences, Yokohama Research Center, Yokohama 227-8502, Japan

* Corresponding author; email: tozaway{at}ccr.ehime-u.ac.jp.

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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[Abstract] [Full Text] [PDF]


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