Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3.
Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein-protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPDeltaN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%.
Bagautdinov
Bagautdin
B
Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
Matsuura
Yoshinori
Y
Bagautdinova
Svetlana
S
Kunishima
Naoki
N
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000235:genetics; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002853:Chromatography, Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D054889:Fatty Acid Synthase, Type II; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization
D002352:Carrier Proteins; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein