Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition.
The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.
Pagan-Rodriguez
Doritza
D
Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, USA.
Zhou
Xiang
X
Simmons
Reiko
R
Bethel
Christopher R
CR
Hujer
Andrea M
AM
Helfand
Marion S
MS
Jin
Zhaoyan
Z
Guo
Baochuan
B
Anderson
Vernon E
VE
Ng
Lily M
LM
Bonomo
Robert A
RA
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000078142:Tazobactam; D014357:Trypsin / Q000494:pharmacology; D001618:beta-Lactamases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012694:Serine; D010397:Penicillanic Acid; D014357:Trypsin; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D000078142:Tazobactam; D005998:Glycine