title: Preliminary crystallographic characterization of BSAP, an extracellular aminopeptidase from Bacillus subtilis.
abstract: 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 A. A full diffraction data set has been collected from such a crystal of the native enzyme (2.2 A resolution, 91.2% completeness, R(merge) = 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 A resolution, 98.8% completeness, R(merge) = 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.
authors.affiliation: Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Vera
authors.initials: V
authors.lastname: Reiland
authors.forename: Yifat
authors.initials: Y
authors.lastname: Fundoiano-Hershcovitz
authors.forename: Gali
authors.initials: G
authors.lastname: Golan
authors.forename: Rotem
authors.initials: R
authors.lastname: Gilboa
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.forename: Gil
authors.initials: G
authors.lastname: Shoham
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D014961:X-Ray Diffraction; D014965:X-Rays; D015032:Zinc / Q000737:chemistry
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D000626:Aminopeptidases; C500523:BSAP extracellular aminopeptidase, Bacillus subtilis; D015032:Zinc