Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose

Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps re quire a divalent cation and the first step has a strong preference for magn esium. Manganese ions are highly unfavourable to the reaction: only 3% resi dual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydrox ybutyrate (AHB), Mn2+ and NADPH. The 1.6 Angstrom resolution electron-densi ty map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) a nd a density corresponding to (phospho)-ADP-ribose instead of the whole NAD P(+). This is one of the few structures of an enzyme complexed with its rea ction product. The structure of this complex was refined to an R factor of 19.3% and an R-free of 22.5%. The overall structure of the enzyme is very s imilar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleav ed and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to th e end of the reaction allowed the proposal of a reaction scheme. Taking thi s result into account, the enzyme was crystallized with Ni2+ and Zn2+, for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/ADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover , mass-spectrometry measurements confirmed the cleavage of nicotinamide.