Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.
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.
Kanno
Takuya
T
Cell-Free Science and Technology Research Center, and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Komatsu
Akira
A
Kasai
Koji
K
Dubouzet
Joseph G
JG
Sakurai
Minako
M
Ikejiri-Kanno
Yasuko
Y
Wakasa
Kyo
K
Tozawa
Yuzuru
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000878:Anthranilate Synthase / Q000235:genetics* / Q000378:metabolism*; D002474:Cell-Free System; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis*
D014364:Tryptophan; D000878:Anthranilate Synthase