In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2.
Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha 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 alpha-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 beta 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 beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta 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.
Kanno
Takuya
T
Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
Kasai
Koji
K
Ikejiri-Kanno
Yasuko
Y
Wakasa
Kyo
K
Tozawa
Yuzuru
Y
D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000494:pharmacology
D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D021122:Protein Subunits; D014364:Tryptophan; D000878:Anthranilate Synthase