caa a22 4500 465824552 CHVBK 20180323112145.0 cr unu---uuuuu 170327e19900801xx s 000 0 eng 10.1007/BF00172550 doi (NATIONALLICENCE)springer-10.1007/BF00172550 Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila [Elektronische Daten] [Ignacio Prieto, Julio Martín, Roberto Arche, Piedad Fernández, Augustín Pérez-Aranda] Summary: 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 k cat 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 k cat and phenylmethylsulphonyl fluoride reactivity, but change the K m.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. Springer-Verlag, 1990 Prieto Ignacio Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut Martín Julio Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Facultad de Ciencias Químicas, 28040, Madrid, Spain aut Arche Roberto Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Facultad de Ciencias Químicas, 28040, Madrid, Spain aut Fernández Piedad Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut Pérez-Aranda Augustín Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut Applied Microbiology and Biotechnology Springer-Verlag 33/5(1990-08-01), 553-559 0175-7598 33:5<553 1990 33 253 https://doi.org/10.1007/BF00172550 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00172550 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Prieto Ignacio Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut NATIONALLICENCE 700 1- Martín Julio Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Facultad de Ciencias Químicas, 28040, Madrid, Spain aut NATIONALLICENCE 700 1- Arche Roberto Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Facultad de Ciencias Químicas, 28040, Madrid, Spain aut NATIONALLICENCE 700 1- Fernández Piedad Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut NATIONALLICENCE 700 1- Pérez-Aranda Augustín Departamento de Investigación, Antibióticos Farma, S. A., Antonio López 109, 28026, Madrid, Spain aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer-Verlag 33/5(1990-08-01), 553-559 0175-7598 33:5<553 1990 33 253 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer