Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli.
Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.
Hosseini-Mazinani
S M
SM
Department of Chemistry II, Faculty of Science, Hokkaido University, Sapporo, Japan.
Nakajima
E
E
Ihara
Y
Y
Kameyama
K Z
KZ
Sugimoto
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D006224:Cricetinae; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial; D006168:Guinea Pigs; D049673:History, 20th Century; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011514:Proteus vulgaris / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D051381:Rats; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D009841:Oligonucleotides; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1