Dataset Information

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of ?-ketothiolase B from Ralstonia eutropha H16.

ABSTRACT: Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. ?-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1?M bis-tris pH 6.5, 0.2?M lithium sulfate at 295?K. X-ray diffraction data were collected to a maximum resolution of 2.3?Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14?Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54?Å(3)?Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

SUBMITTER: Kim EJ

PROVIDER: S-EPMC3944692 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

Kim Eun-Jung EJ Son Hyeoncheol Francis HF Chang Jeong Ho JH Kim Kyung-Jin KJ

Acta crystallographica. Section F, Structural biology communications 20140219 Pt 3

Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, ...[more]

PMID: 24598917

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Project description:beta-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3'-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single- and multiple-deletion mutants were generated to investigate the influence of these beta-ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16Delta2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16Delta2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth beta-ketothiolase or a combination of several of the other beta-ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.

Dataset Information

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of ?-ketothiolase B from Ralstonia eutropha H16.

Publications

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

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