The availability of extremely powerful X-ray sources, as those of ESRF in Grenoble, makes it possible to observe enzymatic reactions, with properly designed equipment, using real time X-ray diffraction. This type of experiments should allow to characterise and understand the key steps of enzymatic reactions, and therefore would lead to better design of inhibitors or enzymes. To understand an enzyme we need to know its native structure. the structures of the various intermediates on the reaction pathway, the kinetic mechanism and the physical organic chemistry of the reaction catalysed. Crystal enzymology offers a direct way to study a wide range of dynamic phenomena associated with the function of biological macromolecules in the crystalline state. Cryo-cooling quenching at certain reaction stages, determined using on line micro- spectrophotometry, or time-resolved diffraction experiments on the catalytic reaction are, to date, the most powerful tools available. We have formed a group of seven laboratories from England, France, Italy, Portugal and Sweden which will design and apply fast X-ray methods for investigating enzyme dynamics in the crystal along reaction pathways. To achieve this goal we propose to undertake a comprehensive and detailed study of a series of related Nitrite Reductases (NiR). The objectives of the project are (i) to characterise biochemically and kinetically several nitrite reductases (NiR) from different organisms, (ii) to determine their 3-D structure, (iii) to develop techniques for triggering and monitoring reactions in their crystals, (iv) to continue the development of fast data collections methods, (v) to apply time-resolved X-ray diffraction techniques in order to determine the three dimensional structures of substrate/intermediate /product complexes of various forms of NiRs, and (vi) in the light of these results, to introduce new catalytic activities into these enzymes by protein engineering. Determination of the structures of several NiR from different organisms and obtention of NiRs with desired functions are prerequisites to reach the main goal. We shall therefore produce, purify and characterise NiRs, and their variants, from 6 different sources.
The NiR system presents an ensemble of favourable characteristics: It is well characterised from structural, biological and kinetical points of view: the structure of oxidised cd1 NiR from Thiosphaera pantotropha (Tp) has already been solved at 1.5A resolution, and that of Pseudomonas aeruginosa (Pa) is likely to be finished soon. The reduced form of Tp-NiR is now available, and demonstrates the great flexibility of this enzyme upon changing the redox state from oxidised to reduced. In the mechanism of reduction of nitrite (N02-) to nitrous oxide (NO), both the substrate binding reaction and the electron transfer can be observed, and preliminary X-ray experiments using cryo-cooling diffraction indicate an important conformational control of the reaction and sufficiently long-lived intermediates to be caught by freesing. Moreover, NiRs are structurally homologous enzymes, performing the same reaction, but exhibiting a wide diversity of structural controls.
Finally, NiRs are part of a chain of enzymes dealing with denitrification, an environmental issue.
Funding SchemeCSC - Cost-sharing contracts
755 91 Uppsala
OX1 3QU Oxford
751 23 Uppsala
2825 Monte De Caparica