Plant and fungal acetohydroxyacid synthases (AHAS): Methods for heterologous expression, kinetic characterization, and structural analysis.

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6), also known as acetolactate synthase, catalyzes the first common step in the biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine. AHAS is present in plants and microorganisms but absent in animals. For this reason, it has become an important target for the development of herbicides and antimicrobial agents. AHAS is the target of five major families of commercial herbicides, which collectively represent one of the most successful groups of herbicides used in modern agriculture. Biochemical, kinetic, and structural studies have revealed that AHAS exhibits a complex catalytic mechanism involving the cofactors thiamine diphosphate (ThDP), flavin adenine dinucleotide (FAD), and Mg2+, as well adopting unique inhibition processes associated with oxidative side reactions that occur under turnover conditions and in the presence of AHAS-inhibiting compounds. Consequently, robust experimental methodologies have been required to investigate AHAS structure, function, and inhibition. Here, we describe a comprehensive set of protocols for the heterologous expression and purification of plant and fungal AHASs, determination of steady-state kinetic parameters, characterization of reversible accumulative inhibition by commercial herbicides, and analysis of herbicide-induced cofactor degradation. Detailed procedures are also provided for monitoring ThDP degradation, FAD redox transitions, and obtaining diffraction-quality crystals suitable for X-ray crystallographic studies. These protocols address several experimental factors that critically influence enzyme activity, including cofactor requirements, lag-phase kinetics, oxygen-dependent side reactions, and inhibitor-induced oxidative processes. Although these methods have been optimized for plant and fungal AHAS enzymes, several of the biochemical and structural approaches described here may also facilitate similar studies of bacterial AHAS enzymes.