title: Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12.
abstract: Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.
authors.affiliation: Abteilung Biochemie, Institut für Entwicklungsbiologie, Tübingen, Federal Republic of Germany.
authors.forename: L C
authors.initials: LC
authors.lastname: Ferreira
authors.forename: U
authors.initials: U
authors.lastname: Schwarz
authors.forename: W
authors.initials: W
authors.lastname: Keck
authors.forename: P
authors.initials: P
authors.lastname: Charlier
authors.forename: O
authors.initials: O
authors.lastname: Dideberg
authors.forename: J M
authors.initials: JM
authors.lastname: Ghuysen
mesh_terms: D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005818:Genetic Engineering; D006602:Hexosyltransferases; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012697:Serine Endopeptidases / Q000235:genetics; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D012697:Serine Endopeptidases