E240V substitution increases catalytic efficiency toward ceftazidime in a new natural TEM-type extended-spectrum beta-lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens clinical isolates.
The aim of this study was to characterize a novel extended-spectrum beta-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149 T182M, in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The bla TEM-149 and bla TEM-149/T182M genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all beta-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149 T182M enzymes, a reduction of the catalytic efficiency for the TEM-149 T182M mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 beta-lactamase.
Perilli
Mariagrazia
M
Department of Sciences and Biomedical Technologies, University of L'Aquila, L'Aquila, Italy.
Celenza
Giuseppe
G
De Santis
Francesca
F
Pellegrini
Cristina
C
Forcella
Chiara
C
Rossolini
Gian Maria
GM
Stefani
Stefania
S
Amicosante
Gianfranco
G
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002384:Catalysis; D002442:Ceftazidime / Q000378:metabolism*; D021902:Enterobacter aerogenes / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012706:Serratia marcescens / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D002442:Ceftazidime; D001618:beta-Lactamases