ReBktB is a beta-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetylCoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 angstrom, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBlaB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of beta-mercaptoethyamine and pantothenic acid Moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K-m, V-max, and K-cat values of 11.58 mu M, 1.5 mu mol/min, and 102.18 s(-1), respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments. (C) 2014 Elsevier Inc. All rights reserved.