Raman crystallographic studies of the intermediates formed by Ser130Gly SHV, a beta-lactamase that confers resistance to clinical inhibitors.
Antibiotic resistance to beta-lactam compounds in Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae is often mediated by beta-lactamase enzymes like TEM and SHV. Previously, a limited number of inhibitors have shown efficacy in combating such bacterial drug resistance. However, many Gram-negative pathogens have evolved inhibitor resistant forms of these hydrolytic enzymes. A single point mutation of the active site residue Ser130 to a Gly in either TEM or SHV results in resistance to amoxicillin and clavulanic acid, an important clinical beta-lactam-beta-lactamase inhibitor combination antibiotic. Previous structural and modeling studies of the S130G mutants of TEM and SHV have shown differences in how these two distinct but closely related enzymes compensate for the loss of the Ser130 residue. In the case of S130G SHV, a structure of tazobactam in the active site has suggested that the inhibitor preferentially assumes a cis-enamine intermediate form when the Ser130 hydroxyl is absent. Raman crystallographic studies of S130G SHV inhibited with tazobactam, sulbactam, clavulanic acid, and 2'-glutaroxy penem sulfone (SA2-13) were performed with the aim of identifying the type and amount of intermediate formed with each drug to understand the role of the S130G mutation in formation of the important enamine intermediates. It is demonstrated that with the exception of sulbactam, each compound forms observable trans-enamine intermediates. For S130G reacted with tazobactam, identical steady state levels of enamine are achieved when compared to those of wild-type (WT) or even deacylation deficient forms of the enzyme. With clavulanic acid, slightly smaller amounts of enamine are observed within the first 30 min of the reaction but are not significantly different than those for tazobactam. Thus, the resistance mutation does not substantially affect the amount of trans-enamine formed with clavulanic acid during the critical early time period of inhibition. This finding has important implications in the design of beta-lactamase inhibitors for drug resistant variants like S130G SHV.
Helfand
Marion S
MS
Research Service and Infectious Diseases Section, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA. marion.helfand@case.edu
Taracila
Magdalena A
MA
Totir
Monica A
MA
Bonomo
Robert A
RA
Buynak
John D
JD
van den Akker
Focco
F
Carey
Paul R
PR
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D019818:Clavulanic Acid / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D013237:Stereoisomerism; D000078142:Tazobactam; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry*; D047090:beta-Lactams / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D019818:Clavulanic Acid; D012694:Serine; D010397:Penicillanic Acid; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D000078142:Tazobactam; D005998:Glycine