Crystal structure of a ternary complex of d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 Å resolution (original) (raw)

D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD -dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low speci®city regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD and 2oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and re®ned to a ®nal R-factor of 19.6% for all measured X-ray re¯ections in the resolution range (I to 1.86 A Ê ). Both NAD and 2-oxoisocaproate were identi®ed in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also de®ned by electron density. Additionally the ®nal model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.