Studies on amino acid replacement and inhibitory activity of a beta-lactamase inhibitory peptide.
An SHV beta-lactamase gene was amplified from a beta-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity of both TEM-1 and SHV beta-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1-C, P1-G, and P1-R, respectively. The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV beta-lactamase were determined. All these beta-lactamase inhibitory peptides could inhibit the activity of both beta-lactamases, while the mutant peptides showed stronger inhibitory activities against TEM-1 beta-lactamase than against SHV beta-lactamase. Inhibition data suggested that P1-A improved the beta-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria-Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study gives us a good candidate for development of novel beta-lactamase inhibitors.
Xie
Liping
L
State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai, China.
Xu
Mingfei
M
Yang
Tao
T
Zhu
Chunbao
C
Zhu
Baoquan
B
Hu
Youjia
Y
D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D006868:Hydrolysis; D010455:Peptides / Q000494:pharmacology*; D065093:beta-Lactamase Inhibitors
D017931:DNA Primers; D010455:Peptides; D065093:beta-Lactamase Inhibitors