THE CRYSTAL-STRUCTURE OF PLANT ACETOHYDROXY ACID ISOMEROREDUCTASE COMPLEXED WITH NADPH, 2 MAGNESIUM-IONS AND A HERBICIDAL TRANSITION-STATE ANALOG DETERMINED AT 1.65-ANGSTROM RESOLUTION

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohy droxy acids into dihydroxy valerates. This reaction is the second in t he synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, th e enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductas e complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 Angstrom resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers relate d by non-crystallographic symmetry. The active site, nested at the int erface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It conta ins two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-bindi ng site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy aci d isomeroreductase sequences and all of these are located around the a ctive site. Finally, a 140 amino acid region, present in plants but ab sent from other species, was found to make up most of the dimerization domain.