Active site mutations of recombinant deacetoxycephalosporin C synthase.
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.
Lee
Hwei-Jen
HJ
Oxford Centre for Molecular Sciences and Dyson Perrins Laboratory, South Parks Road, Oxford, OX1 3QY, UK. hjlee@ndmctsgh.edu.tw
Schofield
Christopher J
CJ
Lloyd
Matthew D
MD
D001665:Binding Sites; D002384:Catalysis; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification
D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D008715:Methionine; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase