title: Overexpression of penicillin V acylase from Streptomyces lavendulae and elucidation of its catalytic residues.
abstract: The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (alpha-subunit) and 60.09 kDA (beta-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobicpocket involved in catalytic activity, including Serbeta1, Hisbeta23, Valbeta70, and Asnbeta272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.
authors.forename: Jesús
authors.initials: J
authors.lastname: Torres-Bacete
authors.forename: Daniel
authors.initials: D
authors.lastname: Hormigo
authors.forename: Raquel
authors.initials: R
authors.lastname: Torres-Gúzman
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
authors.forename: María Pilar
authors.initials: MP
authors.lastname: Castillón
authors.forename: Luis  José
authors.initials: L
authors.lastname: García
authors.forename: Carmen
authors.initials: C
authors.lastname: Acebal
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010399:Penicillin Amidase