A Loop Deletion in the Plant Acetohydroxy Acid Isomeroreductase Homodimer Generates an Active Monomer with Reduced Stability and Altered Magnesium Affinity

Abstract

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

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

“…3). 17 In comparing C-domain association across the four structures, no discernible difference in orientation or interactions could be found. Thus, as the enzyme moves through the different stages of catalysis, dimer association is unaffected.…”

The crystal structure of a bacterial Class II ketol‐acid reductoisomerase: Domain conservation and evolution

“…One notable difference between the two structures is a loop spanning F422–P432 in spinach KARI where there is no corresponding structure in the E. coli protein. This loop in spinach KARI has been shown to be responsible for formation of the homodimer (Wessel et al 1998). Conversely, in E. coli KARI, the region F435–E463 does not have a structural equivalent in the spinach enzyme.…”

“…The spinach KARI asymmetric unit is a tetramer, although the enzyme in solution is a dimer (Wessel et al 1998), with each active site totally contained within a monomer (Biou et al 1997). In contrast, the active site of P. aeruginosa KARI is formed from a pair of monomers and the enzyme is composed of six dimers arranged on the edges of a tetrahedron (Ahn et al 2003).…”

Double Proton Transfer during a Novel Tertiary α-Ketol Rearrangement in Ketol-Acid Reductoisomerase: A Water-Mediated, Metal-Catalyzed, Base-Induced Mechanism

“…Wessel et al measured experimentally the activation energy for the overall reaction in the wild-type acetohydroxy acid isomeroreductase homodimer to be 11.03 kcal/mol; for a monomeric mutant (loop deletion), it was 20.59 kcal/mol. 51 Because Sso-KARI is a thermophilic protein, its activation energy is expected to be significantly higher than those of other KARI variants. 51 Till date, the experimentally measured activation energy for Sso-KARI has not been published yet.…”

“…51 Because Sso-KARI is a thermophilic protein, its activation energy is expected to be significantly higher than those of other KARI variants. 51 Till date, the experimentally measured activation energy for Sso-KARI has not been published yet.…”