Crystal structure and biochemical properties of ReH16_A1887, the 3-ketoacyl-CoA thiolase from Ralstonia eutropha H16.

ReH16_A1887 from Ralstonia eutropha is an enzyme annotated as a 3-ketoacyl-CoA thiolase, and it catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. We determined the crystal structures of ReH16_A1887 in the apo-form and in complex with its CoA substrate. ReH16_A1887 functions as a dimer, and the monomer of ReH16_A1887 comprises three subdomains (I, II, and III). The structural comparison between the apo-form and the CoA-bound form revealed that ReH16_A1887 undergoes a structural change in the lid-subdomain (subdomain III) upon the binding of the CoA substrate. The CoA molecule was stabilized by hydrogen bonding with positively charged residues such as Lys18, Arg210, and Arg217, and residues Thr213 and Gln151 aid its binding as well. At the active site of ReH16_A1887, highly conserved residues such as Cys91, His348, and Cys378 were located near the thiol-group of CoA, indicating that ReH16_A1887 might catalyze the thiolase reaction in a way similar to other thiolases. Moreover, in the vicinity of the covalent nucleophile Cys91, a hydrophobic hole that might serve as a binding site for the acyl-group of 3-ketoacyl-CoA was observed. The residues involved in enzyme catalysis and substrate-binding were further confirmed by site-directed mutagenesis experiments.