Final Report Summary - AEOM (Constructing an artificial enzyme for olefin metathesis)
Strategies to create enzymes with any desired activity could offer tools to streamline synthetic pathways while minimizing environmental impact. Nature has evolved the vast majority of her enzymes from a single architectural motif: the polypeptide. Building on that model, chemists have attempted to create completely new enzymes by co-opting nature's methods for protein evolution, and, alternatively, retooling nature's current set of enzymes for novel tasks. The funding from the Marie Curie program has enabled us to make contributions in both of these areas.
Some of nature's most amazing proteins contain an organometallic prosthetic group. These often highly reactive moieties are shielded from decomposition and off-target reactivity, and funnelled towards the required catalytic process through a refined set of interactions with proximal amino acid side-chains. Such incredible choreography all along the reaction coordinate is humbling to chemists, who are, due to incomplete understanding, still bound to a reductionist approach in reaction development focused on transition state analysis. The recent emergence of molecular biology, particularly recombinant DNA techniques, for the control of protein chemistry, throws open the door for chemists to 'evolve' their own catalysts. The principle is straightforward: take an organometallic core that is catalytically competent and graft it into a suitable protein scaffold. Then modify the protein scaffold using recombinant techniques to optimise any desired reaction parameter.
Our efforts to apply this guiding principle for hybrid catalyst design began with the goal of creating a protein-based catalyst for olefin metathesis. Olefin metathesis is an incredibly powerful reaction that involves the rearrangement of carbon-carbon double bonds. There is no biological counterpart for olefin metathesis, and this would offer the opportunity to carry out metathesis in the biological milieu without affecting endogenous cellular processes. Ultimately we hoped to incorporate a metathesis cofactor into a large capsid protein, lumazine synthase, but we started with a simple monomeric protein to determine first if metathesis in proteins was feasible.
A mutant of the glutaredoxin enzyme that has one free cysteine was expressed in E. coli and covalently linked to a Grubbs-type olefin metathesis catalyst through a judiciously positioned-bromo ester electrophile. A number of olefin metathesis reactions were tested in water to find a suitable probe for catalytic activity. Unfortunately no activity was seen with the protein-catalyst conjugate under all conditions tested; a representative example is shown left. We reasoned that the catalyst was likely binding with the protein scaffold through hydrophobic interactions and was not available for binding with the substrate.
Two experiments support this hypothesis, first the hybrid catalyst was treated with a broad spectrum protease, tryptin, to digest most of the protein leaving the catalyst more exposed, and then subjected to the same conditions for olefin metathesis on the model substrate. Under these conditions a small amount of the ring-closing metathesis product was observed, suggesting the protein deactivates-but does not destroy-the catalyst. Second, if the metathesis reaction is run in the presence of surfactants such as sodium dodecyl sulfate, which would interrupt any cofactor-protein hydrophobic interactions, a small amount of activity is again observed. Current efforts are focused on understanding the nature of the deactivation effect by testing the catalyst in different protein scaffolds with radically different binding pockets.
One important reason for studying the mechanisms of natural enzymatic processes is the inspiration it provides for synthetic chemistry.
Some of nature's most amazing proteins contain an organometallic prosthetic group. These often highly reactive moieties are shielded from decomposition and off-target reactivity, and funnelled towards the required catalytic process through a refined set of interactions with proximal amino acid side-chains. Such incredible choreography all along the reaction coordinate is humbling to chemists, who are, due to incomplete understanding, still bound to a reductionist approach in reaction development focused on transition state analysis. The recent emergence of molecular biology, particularly recombinant DNA techniques, for the control of protein chemistry, throws open the door for chemists to 'evolve' their own catalysts. The principle is straightforward: take an organometallic core that is catalytically competent and graft it into a suitable protein scaffold. Then modify the protein scaffold using recombinant techniques to optimise any desired reaction parameter.
Our efforts to apply this guiding principle for hybrid catalyst design began with the goal of creating a protein-based catalyst for olefin metathesis. Olefin metathesis is an incredibly powerful reaction that involves the rearrangement of carbon-carbon double bonds. There is no biological counterpart for olefin metathesis, and this would offer the opportunity to carry out metathesis in the biological milieu without affecting endogenous cellular processes. Ultimately we hoped to incorporate a metathesis cofactor into a large capsid protein, lumazine synthase, but we started with a simple monomeric protein to determine first if metathesis in proteins was feasible.
A mutant of the glutaredoxin enzyme that has one free cysteine was expressed in E. coli and covalently linked to a Grubbs-type olefin metathesis catalyst through a judiciously positioned-bromo ester electrophile. A number of olefin metathesis reactions were tested in water to find a suitable probe for catalytic activity. Unfortunately no activity was seen with the protein-catalyst conjugate under all conditions tested; a representative example is shown left. We reasoned that the catalyst was likely binding with the protein scaffold through hydrophobic interactions and was not available for binding with the substrate.
Two experiments support this hypothesis, first the hybrid catalyst was treated with a broad spectrum protease, tryptin, to digest most of the protein leaving the catalyst more exposed, and then subjected to the same conditions for olefin metathesis on the model substrate. Under these conditions a small amount of the ring-closing metathesis product was observed, suggesting the protein deactivates-but does not destroy-the catalyst. Second, if the metathesis reaction is run in the presence of surfactants such as sodium dodecyl sulfate, which would interrupt any cofactor-protein hydrophobic interactions, a small amount of activity is again observed. Current efforts are focused on understanding the nature of the deactivation effect by testing the catalyst in different protein scaffolds with radically different binding pockets.
One important reason for studying the mechanisms of natural enzymatic processes is the inspiration it provides for synthetic chemistry.