Biochemical characterization of the THIN-B metallo-beta-lactamase of Janthinobacterium lividum.
The THIN-B metallo-beta-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.
Docquier
Jean-Denis
JD
Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, Siena, Italy.
Lopizzo
Teresa
T
Liberatori
Sabrina
S
Prenna
Manuela
M
Thaller
Maria Cristina
MC
Frère
Jean-Marie
JM
Rossolini
Gian Maria
GM
D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D020560:Proteobacteria / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002511:Cephalosporins; D002614:Chelating Agents; D003470:Culture Media; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014357:Trypsin; C425147:beta-lactamase THIN-B; D001618:beta-Lactamases