7K2W

Crystal structure of CTX-M-14 E166A/K234R Beta-lactamase in complex with hydrolyzed cefotaxime

Summary for 7K2W

• Entry DOI: 10.2210/pdb7k2w/pdb
• Descriptor: Beta-lactamase, CEFOTAXIME, C3' cleaved, open, bound form (3 entities in total)
• Functional Keywords: antibiotic resistance, acyl-enzyme intermediate, hydrolase
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 28082.61

Authors

Lu, S.,Palzkill, T.,Sankaran, B.,Hu, L.,Soeung, V.,Prasad, B.V.V. (deposition date: 2020-09-09, release date: 2020-11-04, Last modification date: 2024-10-16)

Primary citation

Soeung, V.,Lu, S.,Hu, L.,Judge, A.,Sankaran, B.,Prasad, B.V.V.,Palzkill, T.
A drug-resistant beta-lactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency.
J.Biol.Chem., 295:18239-18255, 2020

PubMed Abstract: Lys 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, 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 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 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, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser 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.

PubMed: 33109613
DOI: 10.1074/jbc.RA120.016103

Experimental method

X-RAY DIFFRACTION (1.4 Å)