The extracellular aminopeptidase from Bacillus subtilis (BSAP) has recently been cloned, overexpressed and purified from Escherichia coli. It is a monomer with a molecular weight of 46 425 Da, consisting of 425 amino-acid residues and a double-zinc catalytic centre. The recombinant enzyme was found to be stable for 20 min at 353 K, to function optimally in the pH range 8-9 and to prefer basic and large hydrophobic N-terminal amino acids in peptide and protein substrates. As such, this enzyme can be used as a representative model for structural, functional and mechanistic studies of monomeric double-zinc aminopeptidases, many of which have been found to be involved in medically important biological activities. In this report, the crystallization and preliminary crystallographic characterization of wild-type BSAP are described. Two different crystal forms are reported, of which the hexagonal form H2 is the more suitable for structural study, with average unit-cell dimensions a = b = 226.5, c = 42.8 Å. A full diffraction data set has been collected from such a crystal of the native enzyme (2.2 Å resolution, 91.2% completeness, Rmerge = 7.1%). A multiwavelength anomalous diffraction (MAD) data set was collected on native (zinc-containing) BSAP at three wavelengths around the zinc absorption edge (peak data set at 2.5 Å resolution, 98.8% completeness, Rmerge = 5.3%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. The data are currently being used to obtain crystallographic phasing and to determine the detailed three-dimensional structure of the enzyme.
|Original language||American English|
|Number of pages||6|
|Journal||Acta Crystallographica Section D: Biological Crystallography|
|Issue number||12 II|
|State||Published - Dec 2004|