PDBsum entry 6v8v

Hydrolase

PDB id: 6v8v

Contents

Protein chain

260 a.a.

Ligands

CAZ

Waters ×269

PDB id:

6v8v

Name:

Hydrolase

Title:

Crystal structure of ctx-m-14 e166a/p167s/d240g beta-lactamase in complex with ceftazidime-2

Structure:

Beta-lactamase. Chain: a. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: blactx-m-14, beta-lactamase ctx-m-14, bla, bla ctx-m-14, bla- ctx-m-14a, blactx-m, blactx-m-14a, blactx-m-14b, blactx-m-14c, blactx-m-27b, blatoho-3, blauoe-2, ctx-m-14, am465_01285, am465_06510, am465_23360, apt94_14605, ben53_26220, bet08_34355, bjj90_27545, bk334_27290, boh76_00730, bon63_16015, bon66_01305, bon69_22545, bon71_04040, bon75_10525, bon76_14325, bon81_01055, bon83_15455, bon86_08515, bon91_02075, bon92_04750, bon94_23850,

Resolution:

1.80Å R-factor: 0.151 R-free: 0.189

Authors:

C.A.Brown,L.Hu,B.Sankaran,B.V.V.Prasad,T.G.Palzkill

Key ref:

C.A.Brown et al. (2020). Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance. J Biol Chem, 295, 7376-7390. PubMed id: 32299911 DOI: 10.1074/jbc.RA119.012489

Date:

12-Dec-19 Release date: 22-Apr-20

Protein chain

Q9L5C7  (Q9L5C7_ECOLX) -  Beta-lactamase from Escherichia coli

Seq: 291 a.a.

Struc: 260 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.5.2.6 - beta-lactamase.
• Pathway: Penicillin Biosynthesis and Metabolism
• Reaction: a beta-lactam + H2O = a substituted beta-amino acid
• Cofactor: Zn(2+)

DOI no: 10.1074/jbc.RA119.012489

J Biol Chem 295:7376-7390 (2020)

PubMed id: 32299911

Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance.

C.A.Brown, L.Hu, Z.Sun, M.P.Patel, S.Singh, J.R.Porter, B.Sankaran, B.V.V.Prasad, G.R.Bowman, T.Palzkill.

ABSTRACT

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.