Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii.
The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8% (w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 A. Conversely, crystals grown in 20% (v/v) 2-methyl-2,4-pentanediol, 7.5% (w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 A, beta = 100.25 degrees. Diffraction data were collected in-house to 3.00 and 2.91 A resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 A3 Da(-1), respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code 1dxh) as the search model.
de Las Rivas
Blanca
B
Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain.
Rodríguez
Héctor
H
Angulo
Iván
I
Muñoz
Rosario
R
Mancheño
José M
JM
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D009954:Ornithine Carbamoyltransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D009954:Ornithine Carbamoyltransferase