Interplay between the Enamine and Imine Forms of the Hydrolyzed Imipenem in the Active Sites of Metallo-β-lactamases and in Water Solution

Abstract

Deactivation of the β-lactam antibiotics in the active sites of the β-lactamases is among the main mechanisms of bacterial antibiotic resistance. As drugs of last resort, carbapenems are efficiently hydrolyzed by metallo-β-lactamases, presenting a serious threat to human health. Our study reveals mechanistic aspects of the imipenem hydrolysis by bizinc metallo-β-lactamases, NDM-1 and L1, belonging to the B1 and the B3 subclasses, respectively. The results of QM­(PBE0-D3/6-31G**)/MM simulations show that the enamine product with the protonated nitrogen atom is formed as the major product in NDM-1 and as the only product in the L1 active site. In NDM-1, there is also another reaction pathway that leads to the formation of the (S)-enantiomer of the imine form of the hydrolyzed imipenem; this process occurs with the higher energy barriers. The absence of the second pathway in L1 is due to the different amino acid composition of the active site loop. In L1, the hydrophobic Pro226 residue is located above the pyrroline ring of imipenem that blocks protonation of the carbon atom. Electron density analysis is performed at the stationary points to compare reaction pathways in L1 and NDM-1. Tautomerization from the enamine to the imine form likely happens in solution after the dissociation of the hydrolyzed imipenem from the active site of the enzyme. Classical molecular dynamics simulations of the hydrolyzed imipenem in solution, both with the neutral enamine and the negatively charged N–C2–C3 fragment, demonstrate a huge diversity of conformations. The vast majority of conformations blocks the C3-atom from the side required for the (S)-imine formation upon tautomerization. Thus, according to our calculations, formation of the (R)-imine is more likely. QM­(PBE0-D3/6-31G**)/MM molecular dynamics simulations of the hydrolyzed imipenem with the negatively charged N–C2–C3 fragment followed by the Laplacian bond order analysis demonstrate that the NC2–C3 – resonance structure is the most pronounced that facilitates formation of the imine form. The proposed mechanism of the enzymatic enamine formation and its subsequent tautomerization to the imine form in solution is in agreement with the recent spectroscopic and NMR studies.

Influence of the Active Site Flexibility on the Efficiency of Substrate Activation in the Active Sites of Bi-Zinc Metallo-β-Lactamases

“…This is in line with the analysis of the ES complex: the carbonyl carbon atom of the substrate is more electrophilic due to the tighter and less flexible binding of the imipenem in the L1 active site. These results are in line with the data obtained on potential energy surface [ 8 ]. The energy barrier is lower, and the intermediate stabilization energy is larger in the L1 compared with the NDM-1.…”

“…Machine learning reveals that all geometry parameters, including distance of the nucleophilic attack, coordination bond lengths between the substrate and zinc cations, and C=O and C–N covalent bond lengths in the active site have similar importance for the Fukui index estimates. Gibbs energy profiles calculated for the hydrolysis of the imipenem in the active sites of the L1 and NDM-1 shares the same features with the profiles on the potential energy surface [ 8 ]. Formation of tighter and more rigid active site in the L1 leads to the lower energy barrier and larger extent of the intermediate stabilization relative to the ES complex.…”

“…To do this, we selected two bacterial enzymes related to the antibiotic resistance, metallo-β-lactamases L1 (a B3 metallo-β-lactamase from Stenotrophomonas ( Xanthomonas ) maltophilia ) and NDM-1 (New Delhi metallo-β-lactamase), belonging to the same B class [ 7 ]. The reaction mechanism of the imipenem antibiotic hydrolysis by these two enzymes has already been studied [ 8 ]. The consequence of the elementary steps is the same for both enzymes; still, the energy barrier at the first step is lower for the L1-containing system.…”

Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products

“…Although our data therefore support the accumulation of the carbapenem-derived ( S )-Δ 1 -imine on the active site, as previously suggested ( 22 ), two possible explanations for this exclusive observation present themselves: either the Δ 2 -enamine product rapidly leaves the L1 active site, tautomerizes in solution to the (thermodynamically favored) ( S )-Δ 1 -imine and then rebinds or the ( S )-Δ 1 -imine slowly accumulates in the active site, leaving more slowly than other tautomeric forms/stereoisomers (whose initial production may be preferred kinetically) due to the increased stability of the L1 complex. Recent QM/MM studies ( 61 ) on the L1 reaction pathway suggest that C-2 protonation of an imipenem-derived anionic intermediate was blocked at the active site by Pro227 on the L10 loop ( Fig. S2 ).…”

Celebrating Diversity, Equity, Inclusion, and Respect in Computational and Theoretical Chemistry

“…Krivitskaya and Khrenova used QM/MM approaches to compare mechanistic aspects of imipenem hydrolysis by two important metallo-β-lactamases, namely, NDM-1 and L1 . It was demonstrated that, although the two enzymes share a similar substrate range, they exhibit distinct reaction pathways with the formation of different reaction products.…”