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Cloning, expression, purification, crystallization and X‐ray crystallographic analysis of PhaA from Ralstonia eutropha
Author(s) -
Kim EunJung,
Kim KyungJin
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14022778
Subject(s) - cloning (programming) , crystallization , ralstonia , x ray , crystallography , chemistry , microbiology and biotechnology , biology , biochemistry , gene , physics , organic chemistry , quantum mechanics , computer science , programming language
Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha ( Re PhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl‐CoA molecules to give acetoacetyl‐CoA. Re PhaA was crystallized using the hanging‐drop vapour‐diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris–HCl pH 8.5 and 0.2 M trimethylamine N ‐oxide dihydrate at 295 K. X‐ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P 2 1 , with unit‐cell parameters a = 68.38, b = 105.47, c = 106.91 Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight ( V M ) is 2.3 Å 3 Da −1 , which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular‐replacement method and refinement of the structure is in progress.