Crystal structure and biochemical characterization of a 3-ketoacyl-CoA thiolase from Ralstoniaeutropha H16.
The protein ReH16_B0759 from Ralstoniaeutropha is a 3-ketoacyl-coenzyme A (CoA) thiolase that catalyzes the fourth step of the β-oxidation degradative pathways by converting 3-ketoacyl-CoAto acyl-CoA. The crystal structures of ReH16_B0759 in its apo form and as a complex with its CoA substrate have been determined. Although ReH16_B0759 exhibited an overall structure similar to the ReH16_A1887 isozyme, the proteindoes not make a complex for β-oxidation. Similar to other degradative thiolases, ReH16_B0759 functions as a dimer, and the monomer comprises three subdomains. Unlike ReH16_A1887, a substantial structural change was not observed upon the binding of the CoA substrate in ReH16_B0759. Exceptionally, the Arg220 residue moved about 5.00Å to make room for the binding of the adenosine ring. Several charged residues including Arg220 are involved in the stabilization of CoA through hydrogen bond interactions. At the active site of ReH16_B0759, highly conserved residues such as Cys89, His347, and Cys377 were located near the thiol-group of CoA, suggesting that ReH16_B0759 may catalyze the thiolase reaction in a manner similar to that of other degradative thiolases. The residues involved in substrate binding and enzyme catalysis were further confirmed by site-directed mutagenesis.
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, South Korea.
Jieun
J
Kim
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, South Korea. Electronic address: kkim@knu.ac.kr.
Kyung-Jin
KJ
Kim
D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D020562:Betaproteobacteria / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity
D000102:Acetyl-CoA C-Acyltransferase