Conversion of a DD-peptidase into a β-lactamase by directed evolution under optimized safety conditions

Details

Supervisors

Soumillion, Patrice

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Despite being essential components of life, enzymes remain poorly understood. With the overall aim of understanding key features that drive enzyme evolution and deepening our understanding of the structure-to-function relationship of enzymes, this master's thesis is part of a project investigating the evolution of serine β-lactamases, a family of enzymes responsible for the hydrolysis of β-lactam antibiotics. Β-lactamases are thought to have evolved from an ancient DD-peptidase, a family of enzymes involved in PG remodelling that are inhibited by β-lactams. Among the DD-peptidases, the PBP-A family -cyanobacterial DD-peptidases- represents the group phylogenetically closest to modern serine β-lactamases. Within this family, PBP-A from Thermosynechococcus elongatus was selected as the starting point for our evolutionary study. However, despite numerous directed evolution campaigns, no variant with improved β-lactamase activity has been successfully isolated. Recent research has shown that the expression of PBP-A in E. coli is associated with a fitness cost, likely due to a promiscuous endopeptidase activity on the peptidoglycan, which weakens the cell wall. In this project, we demonstrate that some key point mutations, namely a change in the isoelectric point, the introduction of a disulfide bond and the disruption of a hydrogen bond interaction with the substrate, substantially reduce the toxicity associated with the expression of PBP-A in E. coli. These mutations prevent PBP A from acting on the peptidoglycan while preserving its ability to interact with β-lactams. Starting from this optimized mutant, random mutagenesis led to the emergence of variants with improved β-lactam resistance. Three resistant mutants showing enhanced ampicillin degradation were isolated. However, it remains to be confirmed whether this improved resistance stems from β-lactamase activity. Overall, these mutants appear to be a promising starting point for unlocking the evolutionary pathway for the conversion of PBP-A into a β-lactamase. By exploring this evolutionary process, this project highlights a key features of enzyme evolution and sheds light on the important, but often underestimated, evolutionary constraint imposed by the fitness cost associated with catalytic enzyme conversion.