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

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the α... 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 NH 4 + as the amino donor. The V max and K i 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2004 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/B:PLAN.0000028729.79034.07
Publisher site
See Article on Publisher Site

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 NH 4 + as the amino donor. The V max and K i 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.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 18, 2004

References

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