title: Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis.
abstract: Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their beta-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 A. The estimated Matthews coefficient was 3.23 A3 Da(-1), corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.
authors.forename: Priya
authors.initials: P
authors.lastname: Rathinaswamy
authors.forename: Archana V
authors.initials: AV
authors.lastname: Pundle
authors.forename: Asmita A
authors.initials: AA
authors.lastname: Prabhune
authors.forename: Hepzibah
authors.initials: H
authors.lastname: Sivaraman
authors.forename: James A
authors.initials: JA
authors.lastname: Brannigan
authors.forename: Guy G
authors.initials: GG
authors.lastname: Dodson
authors.forename: C G
authors.initials: CG
authors.lastname: Suresh
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D030562:Databases, Protein; D004058:Diffusion; D005561:Formates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D005561:Formates; D011994:Recombinant Proteins; C030544:formic acid; D010399:Penicillin Amidase