Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16.
ReBktB is a β-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 acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3Å, 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 ReBktB. 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 β-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 μM, 1.5 μmol/min, and 102.18 s(-1), respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.
Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Eun-Jung
EJ
Kim
Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Hyeoncheol Francis
HF
Son
Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea; School of Nono-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea.
Sangwoo
S
Kim
Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Jae-Woo
JW
Ahn
Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea. Electronic address: kkim@knu.ac.kr.
Kyung-Jin
KJ
Kim
D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D020118:Cupriavidus necator / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D054813:Polyhydroxyalkanoates; D000102:Acetyl-CoA C-Acyltransferase
Beta-keto thiolase; Crystal structure; Polyhydroxyalkanoates; Ralstonia eutropha