Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene.
A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.
Saito
Y
Y
Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan.
Fujimura
T
T
Ishii
Y
Y
Noguchi
Y
Y
Miura
T
T
Niwa
M
M
Shimomura
K
K
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship
D002511:Cephalosporins; C025163:cephalosporin C; D000581:Amidohydrolases; C019665:amidase