The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases

Abstract

The interplay of enzyme active site electrostatics and chemical positioning is important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 β-lactamase to TEM-52 toward extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure–function paradigm and noncovalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM β-lactamase antibiotic resistance.

Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of β-Lactamases

“…Samples of TEM·AVB for infrared spectroscopy were prepared by mixing AVB with TEM-S70G, a nucleophile-impaired mutant, which traps the noncovalent complex . To extract the AVB CO’s vibrational peaks from the overwhelming protein background, we synthesized AVB with an isotope-labeled 13 CO (Figures c and S15–S23), which displays the expected isotope redshift of 47 cm –1 with respect to 12 CO (Figure S24 and Table S15).…”

“…We chose TEM-1 β-lactamase, a culprit of antibiotic resistance, as the model enzyme because it has been extensively studied as a target of covalent inhibition. TEM-1 β-lactamase rapidly hydrolyzes penicillin G (PenG), a β-lactam substrate, through a two-step mechanism. , The hydroxy group of S70 attacks PenG’s β-lactam carbonyl (CO), generating an acyl-enzyme (Figure a), which is subsequently deacylated via hydrolysis for a catalytic turnover (Table S1). By contrast, a similar nucleophilic attack on the urea CO of avibactam (AVB) forms a carbamyl-enzyme stable to hydrolysis, thus trapping the enzyme in the covalent complex (Figure b) .…”

Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments

“…This frequency–field dependence can be calibrated by measuring vibrational solvatochromism and calculating average fields using molecular dynamics (MD) simulations, as shown for a carbonyl group in Figure A. This approach has been used with functionally relevant carbonyls to correlate electric fields at enzyme active sites with activation free energies. − While a similar linear frequency–field correlation also applies to nitriles for aprotic solvents, the aforementioned blueshift in protic solvents lies far from this correlation, shown for benzonitrile’s −CN in Figure B. Computational approaches that account for H-bonding frequency shifts are available but are difficult to implement experimentally, , and experiments characterizing H-bonding , have not been used to correct H-bond frequency shifts in order to obtain electric fields.…”

EnzyHTP: A High-Throughput Computational Platform for Enzyme Modeling

“…Alternatively, one could measure kinetic parameters and examine their correlation with the predicted electrostatic stabilization energies in different variants. However, such a correlation might not exist when the enzyme kinetics are significantly influenced by the change of substrate–protein dynamics upon mutation, as recently reported in the study of the evolutionary trajectory of TEM β-lactamases …”