Plant Molecular Biology

, Volume 54, Issue 1, pp 11–23 | Cite as

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the α subunits OASA1 and OASA2

  • Takuya Kanno
  • Koji Kasai
  • Yasuko Ikejiri-Kanno
  • Kyo Wakasa
  • Yuzuru Tozawa

Abstract

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, α and β, and converts chorismate to anthranilate. Two or more AS α-subunit genes have been identified and characterized in several land plants. Although α subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS α-subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS β subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two β subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a β subunit to achieve maximal enzymatic activity even with NH4+ as the amino donor. The Vmax and Ki for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.

anthranilate synthase Oryza sativa rice tryptophan biosynthesis wheat germ cell-free protein synthesis 

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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Takuya Kanno
    • 1
    • 2
    • 4
  • Koji Kasai
    • 2
  • Yasuko Ikejiri-Kanno
    • 1
  • Kyo Wakasa
    • 2
    • 3
  • Yuzuru Tozawa
    • 1
    • 2
    • 4
  1. 1.Cell-Free Science and Technology Research CenterEhime UniversityMatsuyama, EhimeJapan
  2. 2.JST/CREST Plant Functions and Their ControlJapan
  3. 3.National Institute of Crop ScienceTsukuba, IbarakiJapan
  4. 4.Mitsubishi Kagaku Institute of Life SciencesYokohama Research CenterAoba-ku, Yokohama, KanagawaJapan

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