PURIFICATION AND CHARACTERIZATION OF A FUSION PROTEIN OF PLANT ACETOHYDROXY ACID SYNTHASE AND ACETOHYDROXY ACID ISOMEROREDUCTASE

The nucleotide sequence coding for the Arabidopsis thaliana acetohydro xy acid synthase was genetically fused in frame with the nucleotide se quence coding for the Spinacia oleracea acetohydroxy acid isomeroreduc tase and expressed in Escherichia coli, This construction allowed the production of large amounts of soluble fusion protein. The pure chimer ic enzyme exhibits high acetohydroxy acid synthase and acetohydroxy ac id isomeroreductase specific activities, Fusion and native enzymes exh ibit similar K-m values for their substrates and for most cofactors, F urthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increase d. Thus, the chimeric enzyme appears to be a useful tool for the deter mination of kinetic and structural properties of plant acetohydroxy ac id synthase. (C) 1997 Federation of European Biochemical Societies.