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 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.
Wessel
P M
PM
Unité Mixte CNRS/Rhône-Poulenc (UMR 41), Rhône-Poulenc Agrochimie, Lyon, France.
Biou
V
V
Douce
R
R
Dumas
R
R
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D050540:Ketol-Acid Reductoisomerase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D011994:Recombinant Proteins; D012694:Serine; D010649:Phenylalanine; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium