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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 26, Number 6
BKCSDE 26(6)
June 20, 2005 

 
Title
FAD-independent and Herbicide-resistant Mutants of Tobacco Acetohydroxy Acid Synthase
Author
Dung Tien Lea, Jung-Do Choi*
Keywords
Acetohydroxy acid synthase, Sequence motif, Tobacco, Site-directed mutagenesis, Flavoenzyme
Abstract
Acetohydroxy acid synthase catalyzes the first common step in the biosynthesis of branched chain amino acids. AHAS plays two distinct metabolic roles, and is designated as anabolic AHAS and catabolic AHAS, depending on its function. Anabolic AHAS is FAD-dependent, while its catabolic counterpart is not. In this work, a conserved motif was identified in the β-domain of anabolic AHASs, but not in catabolic AHAS (372RFDDR376). In order to determine the functions of this motif, we replaced the motif with the corresponding sequence in FAD-independent AHAS, SPVEY. None of these three mutants (SPV, SPVE, and SPVEY) was detected with bound FAD. However, two of these mutants (SPVE and SPVEY) were active at a low level of specific activity. Although they exhibited pyruvate- and ThDP- dependent characteristics, the activity of the two active mutants appears to be FAD-independent. The SPVEY mutant was completely insensitive to the three tested herbicides, even at extremely high concentrations and is also somewhat more thermolabile than the wild type enzyme. The data provided in this work suggest that the RFDDR motif is a possible determinant of the FAD-dependent and herbicide-resistant properties of tobacco AHAS. The SPVEY mutant appears to exhibit catabolic AHAS-like activity.
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916 - 0
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