Enzyme engineering and evolution
Enzymes are biological catalysts featuring extremely high efficiency, optimised specificity and are often equipped with activity control mechanisms for adapting their rates to the physiological demand. Our research is focused on investigating the biochemical mechanisms underlying these remarkable properties as well as the molecular mechanisms of mutagenesis and selection that led to enzyme emergence and optimisation over the millions of years of evolution.
To this aim, we combine modern molecular biology with classical biochemistry for engineering enzyme variants and studying their properties. We also develop directed evolution strategies for artificially evolving enzymes in the lab by mimicking the Darwinian principle of mutations and selection. In practice, we use random mutagenesis techniques for creating large libraries of enzyme variants that will be expressed in bacteria "(between a million and a billion of individual clones)." Enzyme variants endowed with a desired property are then identified using an appropriate selection or screening strategy and further characterized by classical enzymology and structural biology.
A first project concerns the conversion of a D-Alanyl-D-Alanine peptidase into a beta-lactamase. DD-peptidases are involved in the peptidoglycan biosynthesis and maintenance and are the targets of beta-lactam antibiotics (such as penicillins) with which they form inactive covalent complexes. They are also called PBPs for Penicillin Binding Proteins. On the other hand, beta-lactamases are hydrolytic enzymes degrading beta-lactam antibiotics and conferring resistance to their bacterial host. These two families of serine enzymes share the same fold and it is generally accepted that beta-lactamases have emerged from one or more ancestral PBPs. Although the conversion of a PBP into a beta-lactamase seems a priori straightforward, it has proven to be extremely difficult in the lab. Our group is now exploring a evolutionary trajectory involving the counter intuitive neutralization of the starting peptidase activity before selecting for beta-lactamase. The idea is to keep the active site secure during evolution.
Another project is focused on the evolution of oligomeric enzymes. The idea here is to test the hypothesis that oligomeric enzyme may have an evolutionary advantage over monomeric ones. When a gene duplicates before evolving towards a new function, the parental gene may play an important chaperoning effect if it encodes an oligomeric enzyme. Indeed, in the early stages of evolution, subunits of the parental protein may stabilize an evolving subunit by forming hetero-oligomeric enzymes. This can be described as a kind of “protein sexuality”. In the lab, we are investigating this hypothesis by testing enzyme conversion trajectories allowing or not hetero-oligomerisation. The model system is the conversion of D-malate dehydrogenase into isocitrate dehydrogenase, an activity that can be easily selected in vivo since it restores glutamate prototrophy.
Cyclic peptides and inteins
Besides enzymes, our team is also interested in engineering cyclic peptide molecules with the aim of discovering new drugs or new bioactive compounds. We use the SICLOPPS technology (Split Intein Cyclization of Peptides and ProteinS) for generating large expression libraries of biosynthetic cyclic peptides in bacteria or yeast. Cyclization, performed by the slpicing of a permuted intein, makes the peptide more stable and a better candidate for potential therapeutic use. Using similar directed evolution strategies as for enzymes, we develop selection or screening methods for identifying interesting peptides in these libraries. More specifically, we are looking for (1) peptides acting as inhibitors of carbapenemases, enzymes that are responsible of a major and worldwide health problem; (2) peptides that are destabilizing the outer membrane of Gram negative bacteria for permeating this membrane and making the bacteria more sensitive to existing antibiotics; and (3) peptides that are preventing beta-aggregation of Alzheimer’s peptide Ab in a yeast expression system.