Peter Moody's Research Interests
Protein Crystallography (X-Ray & Neutron)
Peter's Publication list
We study the biochemical mechanisms of enzyme function by determining their three-dimensional structure using crystallography.
The process usually involves
- Identifying the gene coding for target, cloning it into a suitable expression system (with the help of Protex ).
- Purification (often using engineered affinity tags)
- Exploring the conditions in which the enzyme crystal can grow using our robotic dispensing, crystallization and observation suite.
- Preserving the crystals at cryogenic temperatures and exposing them to X-ray or neutron beams and recording the diffraction patterns. The X-rays are either generated in-house or at synchrotrons such as Diamond in Oxfordshire or ESRF in Grenoble. Neutron beams are generated at the nuclear reactor ILL (Grenoble). The diffraction pattern are detected and measured using either CCD or image-plate devices. In the case of X-ray diffraction, the electrons density of the atoms within the protein molecule can be calculated from the diffraction, allowing us to see the three-dimensional structure of the molecule. In the case of neutron diffraction, the nuclei are seen.
Structure and Mechanism of Redox Enzymes (particularly Haem Enzymes)
Our current work focuses on enzymes that use the iron-containing haem group (or heme to our North American friends) and some of the core metabolic enzymes in enteric pathogens.
The work on the haem enzymes concentrates the enzymes Ascorbate Peroxidase, Cytochrome c peroxidase and is in collaboration with Professor Emma Raven of the Chemistry Department.
Ascorbate peroxidase catalyses oxidation of various substrates using H202. Our structures have shown the separate binding sites of ascorbate and aromatic substrates, and the influence of the protein side-chains on binding. This has enabled us to understand the chemistry of electron and proton transfer in these enzymes. We have also engineered a mutant that moves one of the iron coordinating side chains depending on the catalytic cycle.
The W41A mutant of Ascorbate Peroxidse showing the movement of the distal histidine side-chain that coordinates the iron.
Isoniazid is a pro-drug used as one of the few effective treatments for TB. In the pathogen it is activated by a haem peroxidase similar to Ascorbate Peroxidase and Cytochrome c Peroxidase. We have solved the structures of Isoniazid bound to these enzymes to explain how the prodrug is activated and why certain mutants are resistant.
Isoniazid in the active pocket of Ascorbate Peroxidase
Because hydrogen atoms have so few electrons, we have to use neutrons to see them. Thus we are also using neutron diffraction to look at the chemistry of these enzymes.
Nuclear density (blue) and electron density (green) on the distal histidine of Cytochrome c Peroxidase
Recording the spectrum from crystal of Cytochrome c Peroxidase at 100 K
Structure and Mechanism of Core Metabolic Enzymes in Pathogens
Helicobacter pylori and Campylobacter jejueni are the causative organisms of gastric ulcers and most food-born diarrhoea. Their parasitic life-style has allowed them to evolve relatively simple metabolisms that may be prime drug targets. The entire genome of examples of H. pylori and C. jejeuni have been sequenced, and we have determined the activity and 3-dimensional structures of the glycolytic enyzyme glyceraldehyde-3-phosphate dehydrogenase from both of these pathogens. Our studies and those of others suggest that gluconeogenesis rather than glycolysis is important in these organisms.
NADP bound to Helicobacter pylori GAPDH
There is a recently discovered family of acyl transfer enzymes that contains an unusual trimeric structure of left-handed β-helices as a common scaffold. We have solved the structure of SAT (Serine Acetyl Transferase, part of the cysteine biosynthesis pathway, responsible producing O-acetyl serine).
Serine Acetyl Transferase showing the trimeric scaffold, details of how cysteine (a competitive inhibitor) binds