Active site mutations of recombinant deacetoxycephalosporin C synthase.

Journal article

Site-directed mutagenesis of active site residues of deacetoxycephalosporin C synthase active site residues was carried out to investigate their role in catalysis. The following mutations were made and their effects on the conversion of 2-oxoglutarate and the oxidation of penicillin N or G were assessed: M180F, G299N, G300N, Y302S, Y302F/G300A, Y302E, Y302H, and N304A. The Y302S, Y302E, and Y302H mutations reduced 2-oxoglutarate conversions and abolished (<2%) penicillin G oxidation. The Y302F/G300A mutation caused partial uncoupling of penicillin G oxidation from 2-oxoglutarate conversion, but did not uncouple penicillin N oxidation from 2-oxoglutarate conversion. Met-180 is involved in binding 2-oxoglutarate, and the M180F mutation caused uncoupling of 2-oxoglutarate from penicillin oxidation. The N304A mutation apparently enhanced in vitro conversion of penicillin N but had little effect on the oxidation of penicillin G, under standard assay conditions.

Authors: Lee, H; Schofield, C; Lloyd, MD

• Publication status: Published
• Journal: Biochemical and biophysical research communications
• Volume: 292
• Issue: 1
• Pages: 66-70
• Publication date: 2002-03-01
• DOI: 10.1006/bbrc.2002.6620
• EISSN: 1090-2104
• ISSN: 0006-291X
• Language: English
• Keywords: Mutagenesis, Site-Directed; Recombinant Proteins; Binding Sites; Models, Chemical; Oxidation-Reduction; Kinetics; Intramolecular Transferases; Methionine; Mutation; Penicillins; Penicillin-Binding Proteins; Catalysis; Ketoglutaric Acids