Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346.
Citrobacter freundii GN346 produces a class C beta-lactamase exhibiting the substrate profile of a typical cephalosporinase. The structural and promoter regions of the cephalosporinase gene, comprising 1408 nucleotides, were completely sequenced. The amino acid sequence of the mature enzyme, comprising 361 amino acids, and its molecular mass, 39,878 Da, were determined. The active site was confirmed to be Ser-64. The amino acid sequence of the enzyme differs from that of the cephalosporinase of C. freundii OS60 by nine residues. The nucleotide sequence of the promoter region suggests a possible attenuator structure. Lys-67, one of the most conserved residues found in class A and C beta-lactamases and penicillin-binding proteins, was converted into arginine, threonine or glutamic acid through site-directed mutagenesis. The Glu-67 enzyme had lost the catalytic activity and the Thr-67 enzyme only showed a trace of activity. The Arg-67 enzyme, which retained a significant amount of the activity, was purified. The Km values of the Arg-67 enzyme for cephalothin, cephaloridine and benzylpenicillin are 13-19 times those of the wild-type enzyme; the kcat values for the three substrates are 37%, 3%, and 36% those of the wild-type enzyme, respectively.
Tsukamoto
K
K
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Tachibana
K
K
Yamazaki
N
N
Ishii
Y
Y
Ujiie
K
K
Nishida
N
N
Sawai
T
T
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002510:Cephalosporinase / Q000302:isolation & purification; D002954:Citrobacter / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D013912:Threonine; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D005971:Glutamates; D012333:RNA, Messenger; D013912:Threonine; D018698:Glutamic Acid; D001120:Arginine; D002510:Cephalosporinase; D001618:beta-Lactamases; D008239:Lysine