Related Experiment Video
Updated: Jun 23, 2026
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Saturation Mutagenesis and Molecular Modeling: The Impact of Methionine 182 Substitutions on the Stability of
Vitaly G Grigorenko1, Alexandra V Krivitskaya2, Maria G Khrenova1,2
1Chemistry Department, Lomonosov Moscow State University, 119991 Moscow, Russia.
Abstract:
Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.
Insights
The study investigated TEM-1 beta-lactamase variants, finding that mutations at position 182 significantly impact thermal stability and reactivation, not catalytic activity. The M182E variant showed the highest activity, while structural interactions influence reactivation.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- TEM-1 beta-lactamase is a key enzyme conferring resistance to beta-lactam antibiotics in Gram-negative pathogens.
- Emerging TEM-1 variants exhibit broader substrate specificity and resistance to inhibitors due to amino acid substitutions.
- The secondary mutation M182T has become increasingly prevalent, suggesting its significant role in enzyme adaptation.
Purpose of the Study:
- To investigate the impact of single amino acid substitutions at position 182 of TEM-1 beta-lactamase.
- To determine the influence of these substitutions on catalytic properties, thermal stability, and thermoreactivation.
- To elucidate the structural and mechanistic basis for observed variations in enzyme properties.
Main Methods:
- Saturating mutagenesis was performed at position 182 of TEM-1.
- Steady-state kinetic parameters were measured for various TEM-1 M182X variants.
- Thermal stability was assessed by melting temperature and thermoreactivation assays.
- Molecular dynamic simulations were employed to analyze structural effects.
Main Results:
- Steady-state parameters for penicillin, cephalothin, and ceftazidime remained similar across M182X variants.
- Melting temperature and thermoreactivation ability varied significantly among variants, depending on the amino acid at position 182.
- The M182E variant exhibited 1.5-fold higher residual enzymatic activity than wild-type TEM-1.
- Structural analysis revealed the importance of the V159-R65-E177 triad; substitutions disrupting this triad reduced reactivation ability.
Conclusions:
- Single amino acid substitutions at position 182 of TEM-1 primarily affect thermal stability and reactivation, not substrate hydrolysis kinetics.
- The nature of the amino acid at position 182 dictates the extent of changes in thermal properties.
- Specific structural interactions, particularly the V159-R65-E177 triad, are crucial for the thermoreactivation capacity of TEM-1 beta-lactamase.
More Related Videos
Related Concept Videos
01:18Mutations in Microorganisms
01:30Spontaneous and Induced Mutations
01:25Mechanism of Antibiotic Resistance in MRSA
01:25Clinical Significance of Antibiotic Resistance