title: Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.
abstract: Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.
authors.affiliation: Highthroughput Factory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Bagautdin
authors.initials: B
authors.lastname: Bagautdinov
authors.forename: Chizu
authors.initials: C
authors.lastname: Kuroishi
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Sugahara
authors.forename: Naoki
authors.initials: N
authors.lastname: Kunishima
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D008970:Molecular Weight; D010957:Plasmids; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019731:Carbon-Nitrogen Ligases