Refined structure of dienelactone hydrolase at 1.8A˚

Abstract

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8A˚resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the car☐yl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014A˚and the error in atomic co-ordinates is estimated to be 0.15A˚. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the β-ketoadipate pathway found in bacteria and fungi. DLH is an α/β protein containing seven helices and eight strands of β-pleated sheet. A single 4-turn 3 10-helix is seen. The active-site Cys123 resides at the N-terminal end of an α-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The β-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central β-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the γ-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.