A LOOP DELETION IN THE PLANT ACETOHYDROXY ACID ISOMEROREDUCTASE HOMODIMER GENERATES AN ACTIVE MONOMER WITH REDUCED STABILITY AND ALTERED MAGNESIUM AFFINITY

Plant acetohydroxy acid isomeroreductase is a stable homodimer which c atalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction. Since the enzyme exhibits no kinetic coope rativity either for its cofactor (NADPH and magnesium) or for its subs trates, the reason for dimerization of this enzyme was not obvious. Re cently, crystallographic studies [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] revealed that the loop of residues 422-431 plays a major pa rt in the dimer interface. To understand the role of the quaternary st ructure of the enzyme, we have deleted residues 423-430 and substitute d Phe 431 for serine. This mutant was further overproduced in Escheric hia coli, purified to homogeneity, and characterized. Gel filtration a nd thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability. Furthermore, kinetic an d fluorescence experiments showed that the behavior of the monomer wit h respect to magnesium was greatly altered. These results demonstrate the function of the quaternary structure of plant acetohydroxy acid is omeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.