title: Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.
abstract: A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.
authors.affiliation: Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Leticia L
authors.initials: LL
authors.lastname: Torres
authors.forename: Angel
authors.initials: A
authors.lastname: Cantero
authors.forename: Mercedes
authors.initials: M
authors.lastname: del Valle
authors.forename: Anabel
authors.initials: A
authors.lastname: Marina
authors.forename: Fernando
authors.initials: F
authors.lastname: López-Gallego
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.forename: José
authors.initials: J
authors.lastname: Berenguer
authors.forename: Aurelio
authors.initials: A
authors.lastname: Hidalgo
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D010399:Penicillin Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D010406:Penicillins; C065221:penicillin K; D010399:Penicillin Amidase; D010400:Penicillin G