STRUCTURE OF RAT LIVER ISOVALERYL-CoA DEHYDROGENASE. Karen Tiffany, Rosemary Paschke, Jerry Vockley^[[section]], and Jung-Ja Kim , Medical College of Wisconsin, Milwaukee, WI 53226 and ^[[section]]MAYO Medical School, Rochester, MN 55905

Isovaleryl-CoA dehydrogenase (IVD) belongs to a family of acyl-CoA dehydrogenases that catalyze the [[alpha]],ß-dehydrogenation of acyl-CoA thioesters. Although these enzymes share similar sequences, catalytic mechanisms, and structural properties, the catalytic base is not conserved. Determination of the three-dimensional structure of IVD will allow a better understanding of the mechanism of substrate oxidation and the nature of substrate specificity and may reveal why the catalytic base of IVD is located in a different segment of the linear sequence than other acyl-CoA dehydrogenases.

Rat liver IVD, cloned and expressed in E. coli, was crystallized in the orthorhombic space group P2₁2₁2₁ with unit cell parameters a=94.0, b=97.7, and c=181.7. A 2.5 Å resolution data set has been collected on an R-axis image plate detector. The molecular replacement method was employed to solve the phases, using medium chain acyl-CoA dehydrogenase (MCAD) as the search model. A rotation search was carried out using the Patterson search procedure in XPLOR. The highest peak that emerged after Patterson correlation refinement was used for the translation search. Iterative steps of XPLOR programs and manual fitting are being executed to refine the model. The R-factor generated after the first rigid body refinement was 47.7%. After two rounds of positional refinement followed by simulated annealing, the current R-factor is 31.6%. Even at this stage of refinement, it can be confirmed that E254, the proposed catalytic base, is located in the active site of IVD near the C_a-C_ß bond of the substrate.

This work was supported by NIH grant GM29076 (JJPK).