Tags

Type your tag names separated by a space and hit enter

Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.
Plant Physiol. 2005 Aug; 138(4):2260-8.PP

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.

Links

PMC Free PDF

Authors+Show Affiliations

Kanno T
Cell-Free Science and Technology Research Center, and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Komatsu A
No affiliation info available
Kasai K
No affiliation info available
Dubouzet JG
No affiliation info available
Sakurai M
No affiliation info available
Ikejiri-Kanno Y
No affiliation info available
Wakasa K
No affiliation info available
Tozawa Y
No affiliation info available

MeSH

Amino Acid SequenceAmino Acid SubstitutionAnthranilate SynthaseCell-Free SystemGene ExpressionMutagenesis, Site-DirectedOrganisms, Genetically ModifiedOryzaSaccharomyces cerevisiaeSequence Homology, Amino AcidTriticumTryptophan

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16040654

Citation

Kanno, Takuya, et al. "Structure-based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway." Plant Physiology, vol. 138, no. 4, 2005, pp. 2260-8.
Kanno T, Komatsu A, Kasai K, et al. Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. Plant Physiol. 2005;138(4):2260-8.
Kanno, T., Komatsu, A., Kasai, K., Dubouzet, J. G., Sakurai, M., Ikejiri-Kanno, Y., Wakasa, K., & Tozawa, Y. (2005). Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. Plant Physiology, 138(4), 2260-8.
Kanno T, et al. Structure-based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway. Plant Physiol. 2005;138(4):2260-8. PubMed PMID: 16040654.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. AU - Kanno,Takuya, AU - Komatsu,Akira, AU - Kasai,Koji, AU - Dubouzet,Joseph G, AU - Sakurai,Minako, AU - Ikejiri-Kanno,Yasuko, AU - Wakasa,Kyo, AU - Tozawa,Yuzuru, Y1 - 2005/07/22/ PY - 2005/7/26/pubmed PY - 2005/12/24/medline PY - 2005/7/26/entrez SP - 2260 EP - 8 JF - Plant physiology JO - Plant Physiol VL - 138 IS - 4 N2 - 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. SN - 0032-0889 UR - https://www.unboundmedicine.com/medline/citation/16040654/Structure_based_in_vitro_engineering_of_the_anthranilate_synthase_a_metabolic_key_enzyme_in_the_plant_tryptophan_pathway_ DB - PRIME DP - Unbound Medicine ER -