Our research group undertakes the directed evolution of enzymes to gain a better understanding of enzymatic catalysis, to create novel biosynthetic activities and to contribute to the improvement of the strategies for undertaking directed evolution. The complexity of enzyme catalysis, consisting of ligand recognition and exquisite discrimination followed by rapid catalytic turnover, has traditionally been difficult to study. Recent developments in molecular modelling of macromolecules, in structural resolution and in powerful molecular biological methodologies now offer approaches for improving our understanding of protein function. Our research program combines these methodologies specifically to explore the fundamentals of enzyme catalysis. The benefits of better understanding enzyme catalysis are numerous: in our laboratory, we are gaining a better understanding of enzyme-based drug resistances and are modifying enzymes for synthetic applications. We are also examining the linkage between the dynamics of proteins and catalytic function, aided by tools that include protein NMR.Finally, we are contributing to the development of new portable biosensing technologies for clinical and point-of-care applications.
This review presents computational methods that experimentalists can readily use to create smart libraries for enzyme engineering and to obtain insights into protein–substrate complexes. Computational tools have the reputation of being hard to use and inaccurate compared to experimental methods in enzyme engineering, yet they are essential to probe datasets of ever-increasing size and complexity. In recent years, bioinformatics groups have made a huge leap forward in providing user-friendly interfaces and accurate algorithms for experimentalists. These methods guide efficient experimental planning and allow the enzyme engineer to rationalize time and resources. Computational tools nevertheless face challenges in the realm of transient modern technology.
Cytochrome P450s oxidize hydrophobic compounds to increase the body's capacity to eliminate those compounds. The oxidation reactions they catalyze hold great potential for biocatalytic applications. However, there is a need to tailor cytochrome P450s to catalyze specific, industrially-relevant reactions. We are contributing to a joint computational/experimental effort to improve the efficiency of evolution of this class of enzymes.
Beta-lactamases cause bacterial resistance to commonly-prescribed antibiotics such as penicillins, cephamycins, and carbapenems. Beta-lactamases efficiently hydrolyse those antibiotics, inactivating their antibacterial properties. By way of directed evolution, we have determined that these enzymes evolve readily, adapting to new antibiotics. Importantly, these enzymes appear to tolerate important changes in their intrinsic protein motions, with little effect on their catalytic efficiency.More
Dihydrofolate reductases (or DHFRs) are essential in all dividing cells. As a result, they are crucial drug targets in proliferative diseases such as cancer and bacterial infections.
We have evolved human DHFR (hDHFR) to be highly resistant to the chemotherapy drug methotrexate, as a tool to improve the efficiency of gene therapy.
Bacteria are increasingly acquiring a distinct, inherently drug-resistant DHFR (R67 DHFR). We have contributed to understand the properties of this enzyme and have discovered its first inhibitors.More
We are participating in a collaborative effort to make clinical biodectection more readily available. Using a lightweight and portable surface plasmon resonance (SPR) instrument, we are developing highly sensitive protein-based coatings for quantification of diverse analytes directly out of biofluids.More
Transglutaminases catalyze protein cross-linking, by bonding an amine (a protein- or peptide-bound lysine) to the terminal amide of a protein- or peptide-bound glutamine. Transglutaminases are increasingly applied to protein-tagging schemes, where various amine-bearing compounds are linked to proteins, providing modified proteins with new properties.More