Abstract
Cephalosporin acylase (CA) is a recently identified N-terminal hydrolase. It is also a commercially important enzyme in producing 7-aminocephalosporanic acid (7-ACA), a backbone chemical in synthesizing semi-synthetic cephalosporin antibiotics. CA is translated as an inactive single chain precursor, being post-translationally modified into an active enzyme. The post-translational modification takes place in two steps. The first intramolecular autocatalytic proteolysis takes place at one end of the spacer peptide by a nucleophilic Ser or Thr, which in turn becomes a new N-terminal Ser or Thr. The second intermolecular modification cleaves off the other end of the spacer peptide by another CA. Two binary structures in complex with glutaryl-7-ACA (the most favored substrate of CAs) and glutarate (side chain of glutaryl-7-ACA) were determined, and they revealed the detailed interactions of glutaryl-7-ACA with the active site residues (Y. Kim and W. G. J. Hol (2001) Chem. Biol., in press). In this report: 1) we have mutated key active site residues into nonfunctional amino acids, and their roles in catalysis were further analyzed; 2) we performed mutagenesis studies indicating that secondary intermolecular modification is carried out in the same active site where deacylation reaction of CA occurs; and 3) the cleavage site of secondary intermolecular modification by another CA was identified in the spacer peptide using mutational analysis. Finally, a schematic model for intermolecular cleavage of CA is proposed.
Highlights
Cephalosporin acylase (CA)1 is a recently identified N-terminal hydrolase, and it has a distinctive structural motif, the so called “␣---␣” structural pattern (1)
In this report: 1) we have mutated key active site residues into nonfunctional amino acids, and their roles in catalysis were further analyzed; 2) we performed mutagenesis studies indicating that secondary intermolecular modification is carried out in the same active site where deacylation reaction of CA occurs; and 3) the cleavage site of secondary intermolecular modification by another CA was identified in the spacer peptide using mutational analysis
We extend our structural work of CA from Pseudomonas diminuta (CAD) further by site-directed mutagenesis to the active site residues of CAD
Summary
Site-directed Mutagenesis of CAD—Mutants of CAD were constructed by site-directed mutagenesis using a polymerase chain reaction. The mutant CAD gene on the pET24d(ϩ) plasmid was transformed into an Escherichia coli DH5␣ host cell for further subcloning. The mutant CAD gene, of which signal peptide had been removed, was subcloned into E. coli BL21(DE3) using overexpression vector pET24d(ϩ) for protein expression. All DNA manipulations were performed according to standard techniques (15). All mutant genes were sequenced using an automatic DNA sequencer (PerkinElmer Life Sciences) to confirm whether or not the targeted mutations were correctly mutated, and no other changes were incorporated
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