Using light to drive chemistry in natural light-activated enzymes.
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
Until now protein engineering approaches have almost exclusively relied on using Nature's standard 20 amino acid building blocks to produce proteins with optimised properties for a range of different applications. However, the recent successes in using non-natural amino acids to expand upon Nature's genetic code have highlighted the potential to revolutionise our understanding of structure-function relationships in proteins. In particular, this approach is highly attractive for providing novel insights into the mechanisms of enzyme catalysis, which would otherwise prove impossible by using standard protein engineering methods. We are using these methods to study the mechanisms of natural light activated biological enzymes, which are exceptional models for mechanistic analysis and engineering towards application in biotechnology.1
Specifically, the project will focus on an important light-driven enzyme, protochlorophyllide oxidoreductase (POR), which catalyses a key step in the production of chlorophyll. Due to its unique requirement for light the reaction chemistry in POR can be triggered by using very short laser pulses and hence, it has become a very important model system for studying various aspects of enzyme catalysis. It has been proposed that excited state interactions between active site residues in the enzyme and the protochlorophyllide substrate drive the subsequent reaction chemistry, which involves a sequential hydride transfer from NADPH followed by a thermally-activated proton transfer from a conserved Tyr residue. The role of this key Tyr residue in both photochemistry and proton transfer will be investigated by using the non-native amino acid, fluorotyrosine. In addition, the student will examine the architecture of the active site of POR by producing engineered proteins that contain genetically encoded infra-red probes at defined positions throughout the enzyme. A powerful spectroscopic technique, known as 2DIR, will then be used to probe the distance between the IR label and the protochlorophyllide substrate throughout the catalytic cycle. This highly interdisciplinary project is at the cutting edge of enzymology research and will provide the student with expertise in new protein engineering methods, protein expression / purification, biochemical assays and various laser spectroscopy techniques.
Specifically, the project will focus on an important light-driven enzyme, protochlorophyllide oxidoreductase (POR), which catalyses a key step in the production of chlorophyll. Due to its unique requirement for light the reaction chemistry in POR can be triggered by using very short laser pulses and hence, it has become a very important model system for studying various aspects of enzyme catalysis. It has been proposed that excited state interactions between active site residues in the enzyme and the protochlorophyllide substrate drive the subsequent reaction chemistry, which involves a sequential hydride transfer from NADPH followed by a thermally-activated proton transfer from a conserved Tyr residue. The role of this key Tyr residue in both photochemistry and proton transfer will be investigated by using the non-native amino acid, fluorotyrosine. In addition, the student will examine the architecture of the active site of POR by producing engineered proteins that contain genetically encoded infra-red probes at defined positions throughout the enzyme. A powerful spectroscopic technique, known as 2DIR, will then be used to probe the distance between the IR label and the protochlorophyllide substrate throughout the catalytic cycle. This highly interdisciplinary project is at the cutting edge of enzymology research and will provide the student with expertise in new protein engineering methods, protein expression / purification, biochemical assays and various laser spectroscopy techniques.
