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Crystal structure and biochemical characterization of beta-keto thiolase CrossMark B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

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Title
Crystal structure and biochemical characterization of beta-keto thiolase CrossMark B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16
Author
Kim, Eun-JungSon, Hyeoncheol FrancisKim, SangwooAhn, Jae-WooKim, Kyung-Jin
Keywords
Beta-keto thiolase; Crystal structure; Polyhydroxyalkanoates; Ralstonia eutropha
Issue Date
201402
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Citation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, v.444, no.3, pp.365 - 369
Abstract
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 2.3 Å, 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 Km, Vmax, and Kcat 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.
URI
http://scholarworks.unist.ac.kr/handle/201301/4085
DOI
http://dx.doi.org/10.1016/j.bbrc.2014.01.055
ISSN
0006-291X
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