The Physical Origins of Enzyme Evolution: Correlating the Active Site Electric Fields of Antibiotic Resistance along Evolutionary Trajectories in TEM β-Lactamases

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

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Specific phenotypic, genomic, and fitness evolutionary trajectories toward streptomycin resistance induced by pesticide co-stressors in Escherichia coli

ISME Communications ◽

10.1038/s43705-021-00041-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Yue Xing ◽

Xiaoxi Kang ◽

Siwei Zhang ◽

Yujie Men

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Selective Pressure ◽

Fold Increase ◽

Streptomycin Resistance ◽

Evolutionary Stage ◽

Fitness Costs ◽

Evolutionary Trajectories ◽

K 12 ◽

Late Stages

AbstractTo explore how co-occurring non-antibiotic environmental stressors affect evolutionary trajectories toward antibiotic resistance, we exposed susceptible Escherichia coli K-12 populations to environmentally relevant levels of pesticides and streptomycin for 500 generations. The coexposure substantially changed the phenotypic, genotypic, and fitness evolutionary trajectories, resulting in much stronger streptomycin resistance (>15-fold increase) of the populations. Antibiotic target modification mutations in rpsL and rsmG, which emerged and dominated at late stages of evolution, conferred the strong resistance even with less than 1% abundance, while the off-target mutations in nuoG, nuoL, glnE, and yaiW dominated at early stages only led to mild resistance (2.5–6-fold increase). Moreover, the strongly resistant mutants exhibited lower fitness costs even without the selective pressure and had lower minimal selection concentrations than the mildly resistant ones. Removal of the selective pressure did not reverse the strong resistance of coexposed populations at a later evolutionary stage. The findings suggest higher risks of the selection and propagation of strong antibiotic resistance in environments potentially impacted by antibiotics and pesticides.

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Mistranslation can promote the exploration of alternative evolutionary trajectories in enzyme evolution

Journal of Evolutionary Biology ◽

10.1111/jeb.13892 ◽

2021 ◽

Author(s):

Jia Zheng ◽

Sinisa Bratulic ◽

Heidi E. L. Lischer ◽

Andreas Wagner

Keyword(s):

Enzyme Evolution ◽

Evolutionary Trajectories

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Predicting allosteric mutants that increase activity of a major antibiotic resistance enzyme

Chemical Science ◽

10.1039/c7sc02676e ◽

2017 ◽

Vol 8 (9) ◽

pp. 6484-6492 ◽

Cited By ~ 19

Author(s):

M. J. Latallo ◽

G. A. Cortina ◽

S. Faham ◽

R. K. Nakamoto ◽

P. M. Kasson

Keyword(s):

Machine Learning ◽

Antibiotic Resistance ◽

Active Site ◽

Beta Lactamase ◽

Active Site Residues ◽

Conformational Ensembles

Allosteric mutations increasingkcatin a beta lactamase act by changing conformational ensembles of active-site residues identified by machine learning.

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An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases

eLife ◽

10.7554/elife.02304 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 45

Author(s):

Jeffrey I Boucher ◽

Joseph R Jacobowitz ◽

Brian C Beckett ◽

Scott Classen ◽

Douglas L Theobald

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Duplication Event ◽

Atomic Resolution ◽

Enzymatic Function ◽

Evolutionary Trajectories ◽

Strict Specificity ◽

Functional Shift ◽

Lactate Dehydrogenases ◽

Ancestral Protein

Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function.

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Metallo-β-Lactamases: Influence of the Active Site Structure on the Mechanisms of Antibiotic Resistance and Inhibition

Biochemistry (Moscow) ◽

10.1134/s0006297921140030 ◽

2021 ◽

Vol 86 (S1) ◽

pp. S24-S37

Author(s):

Elena O. Levina ◽

Maria G. Khrenova

Keyword(s):

Antibiotic Resistance ◽

Active Site ◽

Site Structure ◽

Active Site Structure

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Thermosensitive PBP2a requires extracellular folding factors PrsA and HtrA1 for Staphylococcus aureus MRSA β-lactam resistance

Communications Biology ◽

10.1038/s42003-019-0667-0 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 6

Author(s):

Mélanie Roch ◽

Emmanuelle Lelong ◽

Olesya O. Panasenko ◽

Roberto Sierra ◽

Adriana Renzoni ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Protein Folding ◽

Antibiotic Resistance ◽

Active Site ◽

Complete Loss ◽

Folding Pathway ◽

Human Pathogen ◽

Clinical Challenge ◽

Aggregation Activity ◽

Horizontal Acquisition

AbstractStaphylococcus aureus is a major human pathogen and represents a clinical challenge because of widespread antibiotic resistance. Methicillin resistant Staphylococcus aureus (MRSA) is particularly problematic and originates by the horizontal acquisition of mecA encoding PBP2a, an extracellular membrane anchored transpeptidase, which confers resistance to β-lactam antibiotics by allosteric gating of its active site channel. Herein, we show that dual disruption of PrsA, a lipoprotein chaperone displaying anti-aggregation activity, together with HtrA1, a membrane anchored chaperone/serine protease, resulted in severe and synergistic attenuation of PBP2a folding that restores sensitivity to β-lactams such as oxacillin. Purified PBP2a has a pronounced unfolding transition initiating at physiological temperatures that leads to irreversible precipitation and complete loss of activity. The concordance of genetic and biochemical data highlights the necessity for extracellular protein folding factors governing MRSA β-lactam resistance. Targeting the PBP2a folding pathway represents a particularly attractive adjuvant strategy to combat antibiotic resistance.

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Antibiotic Resistance Evolution Is Contingent on the Quorum-Sensing Response in Pseudomonas aeruginosa

Molecular Biology and Evolution ◽

10.1093/molbev/msz144 ◽

2019 ◽

Vol 36 (10) ◽

pp. 2238-2251 ◽

Cited By ~ 9

Author(s):

Sara Hernando-Amado ◽

Fernando Sanz-García ◽

José Luis Martínez

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Quorum Sensing ◽

Wild Type ◽

Resistance Mutations ◽

Epistatic Interactions ◽

Adaptive Mutations ◽

Resistance Evolution ◽

Evolutionary Trajectories

Abstract Different works have explored independently the evolution toward antibiotic resistance and the role of eco-adaptive mutations in the adaptation to a new habitat (as the infected host) of bacterial pathogens. However, knowledge about the connection between both processes is still limited. We address this issue by comparing the evolutionary trajectories toward antibiotic resistance of a Pseudomonas aeruginosa lasR defective mutant and its parental wild-type strain, when growing in presence of two ribosome-targeting antibiotics. Quorum-sensing lasR defective mutants are selected in P. aeruginosa populations causing chronic infections. Further, we observed they are also selected in vitro as a first adaptation for growing in culture medium. By using experimental evolution and whole-genome sequencing, we found that the evolutionary trajectories of P. aeruginosa in presence of these antibiotics are different in lasR defective and in wild-type backgrounds, both at the phenotypic and the genotypic levels. Recreation of a set of mutants in both genomic backgrounds (either wild type or lasR defective) allowed us to determine the existence of negative epistatic interactions between lasR and antibiotic resistance determinants. These epistatic interactions could lead to mutual contingency in the evolution of antibiotic resistance when P. aeruginosa colonizes a new habitat in presence of antibiotics. If lasR mutants are selected first, this would constraint antibiotic resistance evolution. Conversely, when resistance mutations (at least those studied in the present work) are selected, lasR mutants may not be selected in presence of antibiotics. These results underlie the importance of contingency and epistatic interactions in modulating antibiotic resistance evolution.

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IDENTIFICATION OF POTENTIAL SALMONELLA TYPHI BETA-LACTAMASE TEM 1 INHIBITORS USING PEPTIDOMIMETICS, VIRTUAL SCREENING, AND MOLECULAR DYNAMICS SIMULATIONS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i1.21520 ◽

2018 ◽

Vol 10 (1) ◽

pp. 91

Author(s):

Rakesh K. R. Pandit ◽

Dinesh Gupta ◽

Tapan K. Mukherjee

Keyword(s):

Molecular Dynamics ◽

Antibiotic Resistance ◽

Virtual Screening ◽

Molecular Dynamics Simulations ◽

Active Site ◽

Beta Lactamase ◽

Ramachandran Plot ◽

Small Peptide ◽

In Silico Docking ◽

Dynamics Simulations

Objective: The purpose of this study was to identify a potential peptidomimetic S. typhi Beta-lactamase TEM 1 inhibitor to tackle the antibiotic resistance among S. typhi.Methods: The potential peptidomimetic inhibitor was identified by in silico docking of the small peptide WFRKQLKW with S. typhi Beta-lactamase TEM 1. The 3D coordinate geometry of the residues of small peptide interacting with the active site of the receptor was generated and mimics were identified using PEP: MMs: MIMIC server. All the identified mimics were docked at the active site of the receptor using Autodock 4.2 and the best-docked complex was selected on the basis of binding energy and number of H-bonds. The complex was then subjected to molecular dynamics simulations of 30 ns using AMBER 12 software package. The stereochemical stability of the Beta-lactamase TEM 1-WFRKQLKW complex was estimated with the help of Ramachandran plot using PROCHECK tool.Results: In the present study, a new potential peptidomimetic inhibitor (ZINC05839264) of Beta-lactamase TEM 1 has been identified based on antimicrobial peptide WFRKQLKW by virtual screening of the MMsINC database. The docking and molecular simulation studies revealed that the mimic binds more tightly to the active site of the receptor than the peptide. The Ramachandran plot also shows that the Beta-lactamase TEM 1-mimic complex is stereo chemically more stable than Beta-lactamase TEM 1-WFRKQLKW complex as more number of residues (93.6%) are falling under the core region of the plot in case of the former.Conclusion: The study shows that the peptidomimetic compound can act as a potential inhibitor of S. typhi Beta-lactamase TEM 1 and further it can be developed into more effective therapeutic to tackle the problem of antibiotic resistance.

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