Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis

Abstract

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.

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg2+ and NADPH Binding as Revealed by Two Crystal Structures

“…For rice KARI, the K m value for Mg 2+ is 7.2 μM, 5 while for spinach KARI, K d and K m values for Mg 2+ are 5 μM and 6 μM, respectively. 6 Curiously, the K m value for Mg 2+ for plant KARI is extremely low, yet for the Escherichia coli enzyme with a similar constellation of active-site residues, the K m is 450 μM. 7 The catalytic mechanism for spinach KARI is ordered with binding of either Mg 2+ or NADPH, initially and independently, to the free enzyme, followed by substrate binding.…”

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65Aresolution

“…The spinach enzyme was shown to be a homodimer of 114 kDa (Dumas et al ., 1992), and its cDNA was used to overexpress the enzyme in Escherichia coli (Dumas et al ., 1992) and to produce mutants by site‐directed mutagenesis (Dumas et al ., 1995). An alignment of all known amino acid sequences from acetohydroxy acid isomeroreductases suggested regions of the enzyme that might play a role in the reaction.…”

Purified RecombinantEscherichia coliKetol-Acid Reductoisomerase Is Unsuitable for Use in a Coupled Assay of Acetohydroxyacid Synthase Activity due to an Unexpected Side Reaction

“…1B) involves migration of the alkyl group of the 2-aceto-2-hydroxyacid to form a 2-keto-3-hydroxyacid that is then reduced by NADPH to a 2,3-dihydroxyacid. The enzyme requires Mg 2ϩ as an obligatory cofactor (8). An alternative continuous assay for AHAS might be devised by coupling the KARI reaction to that catalyzed by AHAS.…”