Multiple Substitutions at Ambler Position 244 in SHV Provide Insight Into Importance of Arg244 in Inhibitor and Substrate Binding

Abstract

Inhibitor resistant class A beta-lactamases are an emerging threat to treatment of gram negative infections. Based on clinical isolates of inhibitor (clavulanate) resistant TEM enzymes with mutations at Ambler position R244, we used site-saturation mutagenesis to explore the importance of this residue in substrate and inhibitor binding in SHV. 16 mutants had increased Minimum Inhibitory Concentration (MIC) values to ampicillin/clavulanate. In contrast, all mutants had decreased MIC values to ampicillin and cephaloridine. Kinetic studies on variants Arg244Ser, -Gln, and -Glu showed a decrease in affinity for both inhibitor and substrates (60–1000 fold lower than SHV-1). Unexpectedly, the kinact values of the mutants were all elevated, arguing against a mechanistic basis for resistance. Employing mass spectrometry, we showed a large portion of the Arg244Ser enzyme is inactivated and multiple products are formed after 15 minutes incubation (1000:1 inhibitor:enzyme ratio). In conclusion, Arg244 is essential for both antibiotic and inhibitor binding. This finding allows us to predict future development of inhibitor resistant enzymes and provides novel insight into the development of future antimicrobials and treatment strategies. Supported by NIH (1R01 A1063517-01), the Veterans Administration Medical Center Merit Review Program, NIH T32 GM07250 and the Case Medical Scientist Training Program.