Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila.
Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant kcat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the kcat and phenylmethylsulphonyl fluoride reactivity, but change the Km. The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.
Departamento de Investigación, Antibióticos Farma, Madrid, Spain.
I
I
Prieto
J
J
Martín
R
R
Arche
P
P
Fernández
A
A
Pérez-Aranda
J L
JL
Barbero
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity / Q000235:genetics
D004269:DNA, Bacterial; D010406:Penicillins; D008715:Methionine; D010399:Penicillin Amidase