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Dynamical Behavior of ?-Lactamases and Penicillin- Binding Proteins in Different Functional States and Its Potential Role in Evolution

ABSTRACT: ?-Lactamases are enzymes produced by bacteria to hydrolyze ?-lactam-based antibiotics, and pose serious threat to public health through related antibiotic resistance. Class A ?-lactamases are structurally and functionally related to penicillin-binding proteins (PBPs). Despite the extensive studies of the structures, catalytic mechanisms and dynamics of both ?-lactamases and PBPs, the potentially different dynamical behaviors of these proteins in different functional states still remain elusive in general. In this study, four evolutionarily related proteins, including TEM-1 and TOHO-1 as class A ?-lactamases, PBP-A and DD-transpeptidase as two PBPs, are subjected to molecular dynamics simulations and various analyses to characterize their dynamical behaviors in different functional states. Penicillin G and its ring opening product serve as common ligands for these four proteins of interest. The dynamic analyses of overall structures, the active sites with penicillin G, and three catalytically important residues commonly shared by all four proteins reveal unexpected cross similarities between Class A ?-lactamases and PBPs. These findings shed light on both the hidden relations among dynamical behaviors of these proteins and the functional and evolutionary relations among class A ?-lactamases and PBPs.

SUBMITTER: Wang F

PROVIDER: S-EPMC7514474 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Project description:The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Project description:Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C-N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS.

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Dynamical Behavior of ?-Lactamases and Penicillin- Binding Proteins in Different Functional States and Its Potential Role in Evolution

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