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β-Lactamases are one of the primary enzymes responsible for antibiotic resistance and have existed for billions of years. The structural differences between a modern class A TEM-1 β-lactamase compared to a sequentially reconstructed Gram-negative bacteria β- lactamase are minor. Despite the similar structures and mechanisms, there are different functions between the two enzymes. We recently identified differences in dynamics effects that result from evolutionary changes that could potentially account for the increase in substrate specificity and catalytic rate. In this study, we used transition path sampling-based calculations of free energies to identify how evolutionary changes found between an ancestral β-lactamase, and its extant counterpart TEM-1 β-lactamase affect rate. 

β-Lactamases are a class of well-studied enzymes that are known to have existed since billions of years ago, starting as a defense mechanism to stave off competitors and are now enzymes responsible for antibiotic resistance. Using ancestral sequence reconstruction, it is possible to study the crystal structure of a laboratory resurrected 2−3 billion year-old β-lactamase. Comparing the ancestral enzyme to its modern counterpart, a TEM-1 β-lactamase, the structural changes are minor, and it is probable that dynamic effects play an important role in the evolution of function. We used molecular dynamics simulations and employed transition path sampling methods to identify the presence of rate-enhancing dynamics at the femtosecond level in both systems, found that these fast motions are more efficiently coordinated in the modern enzyme, and examined how specific dynamics can pinpoint evolutionary effects that are essential for improving enzymatic catalysis.