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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Insights into structure and activity relationship of clinically mutated PER1 and PER2 class A β-lactamase enzymes
Vidhu Agarwal1, Tara Chand Yadav2, Akhilesh Tiwari1
1Department of Applied Sciences, Indian Institute of Information Technology, Jhalwa, Allahabad, India.
Abstract:
PER1 and PER2 are among the class A β-lactamase enzymes, which have evolved clinically to form antibiotic resistance and have significantly expanded their spectrum of activity. Hence, there is a need to study the clinical mutation responsible for such β-lactamase mediated antibiotic resistance. Alterations in catalytic centre and Ω-loop structure could be the cause of antibiotic resistance in these β-lactamase enzymes. Structural and functional alterations are caused due to mutations on or near the catalytic centre, which results in active site plasticity and are responsible for its expanded spectrum of activity in these class A β-lactamase enzymes. Multiple sequence alignment, structure, kinetic, molecular docking, MMGBSA and molecular dynamic simulation comparisons were done on 38 clinically mutated and wild class A β-lactamase enzymes. This work shows that PER1 and PER2 enzymes contains most unique mutations and have altered Ω-loop structure, which could be responsible for altering the structure-activity relationship and extending the spectrum of activity of these enzymes. Alterations in molecular docking, MMGBSA, kinetic values reveals the modification in the binding and activity of these clinically mutated enzymes with antibiotics. Further, the cause of these alterations can be revealed by active site interactions and H-bonding pattern of these enzymes with antibiotics. Met69Gln, Glu104Thr, Tyr105Trp, Met129His, Pro167Ala, Glu168Gln, Asn170His, Ile173Asp and Asp176Gln mutations were uniquely found in PER1 and PER2 enzymes. These mutations occurs at catalytic important residues and results in altered interactions with β-lactam antibiotics. Hence, these mutations could be responsible for altering the structure-activity of PER1 and PER2 enzymes.Communicated by Ramaswamy H. Sarma.
Insights
Unique mutations in PER1 and PER2 enzymes, specifically in the Ω-loop, alter their structure and antibiotic resistance. These findings explain the expanded activity spectrum of these class A beta-lactamase enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Class A beta-lactamase enzymes, including PER1 and PER2, are critical in the clinical evolution of antibiotic resistance.
- Mutations in the catalytic center and Ω-loop are implicated in the expanded activity spectrum and resistance mechanisms of these enzymes.
Purpose of the Study:
- To investigate the specific clinical mutations in PER1 and PER2 enzymes responsible for antibiotic resistance.
- To understand how structural and functional alterations, particularly in the Ω-loop, contribute to the expanded activity spectrum of these class A beta-lactamase enzymes.
Main Methods:
- Comparative analysis of 38 clinically mutated and wild-type class A beta-lactamase enzymes using multiple sequence alignment, structural, kinetic, molecular docking, MMGBSA, and molecular dynamic simulations.
- Identification of unique mutations in PER1 and PER2 enzymes and analysis of their impact on active site interactions and H-bonding patterns with beta-lactam antibiotics.
Main Results:
- PER1 and PER2 enzymes exhibit unique mutations, notably an altered Ω-loop structure, correlating with altered structure-activity relationships.
- Mutations such as Met69Gln, Glu104Thr, Tyr105Trp, Met129His, Pro167Ala, Glu168Gln, Asn170His, Ile173Asp, and Asp176Gln were uniquely identified in PER1 and PER2.
- These mutations occur at catalytically important residues, leading to altered interactions with beta-lactam antibiotics, as evidenced by changes in molecular docking, MMGBSA, and kinetic values.
Conclusions:
- The unique mutations and altered Ω-loop structure in PER1 and PER2 enzymes are key drivers of their expanded activity spectrum and antibiotic resistance.
- These findings provide insights into the molecular mechanisms underlying beta-lactamase-mediated antibiotic resistance and can inform the development of new therapeutic strategies.
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