CRYSTAL-STRUCTURE OF A TERNARY COMPLEX OF D-2-HYDROXYISOCAPROATE DEHYDROGENASE FROM LACTOBACILLUS-CASEI, NAD(-OXOISOCAPROATE AT 1.9 ANGSTROM RESOLUTION() AND 2)

D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus case i is a homodimer with 333 amino acids and a molecular mass of 37 kDa p er subunit. The enzyme belongs to the protein family of NAD(+)-depende nt 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 specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. He xagonal crystals of recombinant D-HicDH in the presence of NAD(+) and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium s ulphate as precipitating agent. The structure of these crystals was so lved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 Angstrom). Both NAD(+) and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, h owever, competes with a sulphate ion, which is also defined by electro n density. Additionally the final model contains 182 water molecules a nd a second sulphate ion. The binding of both an in vitro substrate an d the natural cosubstrate in the active site provides substantial insi ght into the catalytic mechanism and allows us to assess previously pu blished active site models for this enzyme family, in particular the t wo most controversial points, the role of the conserved Arg234 and sub strate binding. Furthermore the overall topology and details of the D- HicDH structure are described, discussed against the background of hom ologous structures and compared with one closely and one distantly rel ated protein. (C) 1997 Academic Press Limited.