Solution structure of 3-oxo-Delta(5)-steroid isomerase

The three-dimensional structure of the enzyme 3-oxo-Delta(5)-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr(14) (general acid) and Asp(38) (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp(99)) is also located in the active site adjacent to Tyr(14), and kinetic and binding studies of the Asp(99) to Ala mutant demonstrate that Asp(99) contributes to catalysis by stabilizing the intermediate.