Download MendeleyA drug-resistant beta-lactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency.
Soeung, V., Lu, S., Hu, L., Judge, A., Sankaran, B., Prasad, B.V.V., Palzkill, T.(2020) J Biol Chem 295: 18239-18255
- PubMed: 33109613
- DOI: https://doi.org/10.1074/jbc.RA120.016103
- Primary Citation of Related Structures:
7K2W, 7K2X, 7K2Y - PubMed Abstract:
Lys 234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser 130 , it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase-mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys 234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in k cat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser 130 , and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser 130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.
Organizational Affiliation:
Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, Texas, USA.
