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Journal of Biochemistry 2005 138(1):35-40; doi:10.1093/jb/mvi099
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© 2005 The Japanese Biochemical Society

Regular Paper

Roles of Three Well-Conserved Arginine Residues in Mediating the Catalytic Activity of Tobacco Acetohydroxy Acid Synthase

Dung Tien Le1,*, Moon-Young Yoon2, Young Tae Kim3 and Jung-Do Choi1,{dagger}

1 School of Life Sciences, Chungbuk National University, Cheongju 361-763, Korea; 2 Department of Chemistry, Hanyang University, Seoul 133-791, Korea; and 3 Department of Microbiology, Pukyong National University, Busan 608-737, Korea

{dagger} To whom correspondence should be addressed. Tel: +82-43-261-2308, Fax: +82-43-267-2306, E-mail: jdchoi{at}chungbuk.ac.kr

Acetohydroxy acid synthase (AHAS, EC 2.2.1.6; also known as acetolactate synthase, ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. AHAS is the target of several classes of herbicides. In the present study, the role of three well-conserved arginine residues (R141, R372, and R376) in tobacco AHAS was determined by site-directed mutagenesis. The mutated enzymes, referred to as R141A, R141F, and R376F, were inactive and unable to bind to the cofactor, FAD. The inactive mutants had the same secondary structure as that of the wild type. The mutants R141K, R372F, and R376K exhibited much lower specific activities than the wild type, and moderate resistance to herbicides such as Londax, Cadre, and/or TP. The mutation R141K showed a strong reduction in activation efficiency by ThDP, while the mutations R372K and R376K showed a strong reductions in activation efficiency by FAD in comparison to the wild type enzyme. Taking into account the data presented here and the homology model constructed previously [Le et al. (2004) Biochem. Biophys. Res. Commun. 317, 930–938], it is suggested that the three amino acid residues studied (R141, R372, and R376) are located essentially at the enzyme active site, and, furthermore, that residues R372 and R376 are possibly responsible for the binding of the enzyme to FAD.

* Present address: The Microbial Engineering Laboratory, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-0856.


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