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. 2022 Dec 7;144(48):22289-22294.
doi: 10.1021/jacs.2c10791. Epub 2022 Nov 18.

β-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields

Zhe Ji  1 , Steven G Boxer  1
Affiliations

β-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields

Zhe Ji et al. J Am Chem Soc. .

Abstract

A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM β-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP-PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site. Conversely, the same linkage in the descendant TEM-PenG experiences large electric fields that stabilize the more charge-separated transition state and thus lower the free energy barrier to hydrolysis. Specifically, the electric fields were improved from -59 to -140 MV/cm in an ancient evolution dating back billions of years, contributing 5 orders of magnitude rate acceleration. This trend continues today in the nullification of newly developed antibiotic drugs. The fast linkage hydrolysis acquired from evolution is counteracted by the upgrade of PenG to Avb whose linkage escapes from the hydrolysis by returning to a low-field environment. Using the framework of electrostatic catalysis, the electric field, an observable from vibrational spectroscopy, provides a unifying physical metric to understand protein evolution and to guide the design of covalent drugs.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Hydrolysis of carbonyl linkages in protein covalent adducts. (A) Mechanism of TEM–PenG hydrolysis. The ester linkage is attacked by a water deprotonated by E166 forming an oxyanion intermediate, which quickly releases penicilloic acid as the product. (B) The rate of hydrolysis determines whether the attached molecule is a covalent inhibitor or a substrate. PBP-A hydrolyzes the ester linkage slowly with a nonpolar L158 at the site of the basic E166 in TEM-1. The hydrolysis is not much improved with a L158E mutation. The protein evolution from PBP-A to TEM-1 improved the linkage hydrolysis, turning PenG from an inhibitor to a substrate. The development of Avb is the reverse process, where the impaired linkage hydrolysis makes Avb a covalent drug. (C) Model of electrostatic catalysis. The electric fields produced by two H-bonds stabilize the transition state more than the reactant because the transition state experiences more charge separation along the C=O bond.
Figure 2.
Figure 2.
Backbone amide H-bonds impose electric fields onto the linkage C=O. (A) Mutational perturbation of the H-bond donated by the A237 backbone amide in TEM. A hydroxy acid counterpart of tyrosine in A237Y was incorporated into the mutant A237Ye using amber suppression, and its backbone ester eliminates one key H-bond to the C=O of substrates or inhibitors. Note that the phenol side chain of A237Ye points away from the binding site (Figure S2). (B) IR absorption spectra displaying the vibrational peaks of the linkage C=O in WT and A237 mutants of TEM−PenG. TEM–PenG was trapped using the E166N mutation. The top electric field axis is mapped from the bottom frequency axis according to ref , ν¯=0.68F+1749.4. ν¯ is the wavenumber (cm−1) of the C=O vibrations, and F is the magnitude of electric fields (MV/cm) projected on the C=O. (C) Overlay of the crystal structure of PBP–PenG (PDB: 2J8Y) and TEME166N–PenG (PDB: 1FQG) by aligning their S61 and S70, respectively, and overlay of the crystal structure of TEME166N–PenG and TEM–Avb (PDB: 8DE0) by aligning their S70. (D) Electric field magnitudes mapped from the vibrational peaks of the linkage C=O in PBP–PenG, TEME166N–PenG, and TEM–Avb.
Figure 3.
Figure 3.
Electrostatic catalysis in the competition between protein evolution and drug development. (A) Plots of free energy barrier of linkage hydrolysis against the largest electric field experienced by the linkage C=O in TEM–PenG (WT and the A237 mutants), PBP–PenG (WT and L158E), and TEM–Avb (WT). The linear correlations for TEM–PenG and TEM–Avb are ΔG‡ = 1.7F + 24.3 (green solid line) and ΔG‡ = 1.4F + 33.7 (blue line), respectively, with ΔG‡ and F in units of kcal/mol and kcal/mol/D, respectively. The difference in intercepts ΔΔGΔF‡=9.4 kcal/mol represents by how much the linkage with Avb is more inert to hydrolysis than the linkage with PenG when there are no stabilizing electric fields. Four comparisons are highlighted: ①, WT PBP–PenG → L158E PBP–PenG; ②, L158E PBP–PenG → WT TEM–PenG; ③, part of WT TEM–PenG → WT TEM–Avb that is due to the change in intrinsic reactivity; ④, part of WT TEM–PenG → WT TEM–Avb that is electrostatic in origin. The green dashed line represents a hypothetical covalent adduct having TEM–PenG’s reaction difference dipole but TEM–Avb’s intrinsic reactivity. (B) Expansion of panel (A) showing the path’s protein evolution and drug development take in leveraging electrostatic catalysis (along slopes) and modulating intrinsic reactivities (vertical). ⑤: TEM-1–Ctx → TEM-52–Ctx. The solid lines connect experimental datapoints, while the dashed lines (⑥ and ⑦) closing the cycle indicate possible paths that can/will be taken for drug development and protein evolution in the future.
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