Alignment of procaryotic and plant acetohydroxy acid isomeroreductase (EC 1.1.1.86) reveals five conserved regions designated domains I, II, III, IV, and V. Domain I has been previously proposed to correspond to the NADPH-binding site [Dumas et al. (1991) Biochem. J. 277, 469-475] and domain III to a putative magnesium-binding site [Sista & Bowman (1992) Gene 120, 115-118]. The binding and the function of this cation are of particular importance. First, Mg2+ is essential for the two-step reaction catalyzed by this enzyme: an isomerization followed by an NADPH-dependent reduction. Second, the plant acetohydroxy acid isomeroreductase exhibits Kd and Km values for Mg2+ of 5 microM and 6 microM, respectively. Such values correspond to the strongest affinity known between an enzyme and the metal ion. To determine if domain III of acetohydroxy acid isomeroreductase is effectively involved in magnesium binding, and with the goal to assign a function to the other conserved domains, site-directed mutagenesis was performed on each charged or polar conserved amino acids of domains II-V of the spinach acetohydroxy acid isomeroreductase. The results demonstrate that mutation of each of these amino acids leads to a partial or complete inactivation of enzyme activity. Steady-state kinetic analysis and equilibrium binding experiments show that both domains III and IV are directly involved in the binding of magnesium. Also, they suggest that magnesium bound to domain III plays a role in the reductive half-reaction, whereas, magnesium bound to domain IV is involved in the isomerization half-reaction.