Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam.
Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.
Zervosen
A
A
Centre for Protein Engineering, University of Liège, Belgium.
Valladares
M H
MH
Devreese
B
B
Prosperi-Meys
C
C
Adolph
H W
HW
Mercuri
P S
PS
Vanhove
M
M
Amicosante
G
G
van Beeumen
J
J
Frère
J M
JM
Galleni
M
M
D016980:Aeromonas hydrophila / Q000201:enzymology*; D001426:Bacterial Proteins; D002513:Cephamycins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D009070:Moxalactam / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D001426:Bacterial Proteins; D002513:Cephamycins; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C082063:cphA protein, Aeromonas hydrophila; D009070:Moxalactam