To freeze or not to freeze: insights into enzyme crystal structures at room temperature

What is it about?

Urocanate reductase (UrdA) is a bacterial enzyme that facilitates the production of imidazole propionate (ImP) and can be found in human gut microbiota. ImP has been linked to several human diseases, including type 2 diabetes. We have previously determined 3D molecular structures of the UrdA using cryogenic X-ray crystallography, which provided structural details of its active site and an insight into enzymatic mechanism. We showed how an important catalytic arginine residue is changing its position in the urocanate versus ImP bound states. In this study we further investigate the UrdA active site by using room-temperature X-ray crystallography. While both methods, cryogenic and room-temperature crystallography use protein crystals for X-ray data collection, applying cryogenic temperatures is known to sometimes mask structural features of the protein, which is in particular important when it comes to biological interpretations. The data collected at room-temperature in presence and absence of sulfate ions revealed more details in the dynamics of the catalytic arginine. Interestingly, we show that in this case the biologically relevant conformation of the arginine is easier captured in cryogenic crystal conditions. We further demonstrate that a sulfate or phosphate ion affects the position of the arginine and stabilizes it in the substrate like conformation.

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Why is it important?

When human gut microbiota contains bacteria with UrdA activity resulting in increased levels of ImP this may contribute to the development of various health issues, such like type 2 diabetes and cardiovascular diseases. A possible therapeutic strategy would be to inhibit the bacterial UrdA by designing molecules binding to the enzyme and in this way blocking the production of ImP. Therefore, the detailed molecular studies of UrdA are necessary to enable drug design. Besides that, our study is relevant for the general use of X-ray crystallography as it demonstrates that the use of both X-ray data collection at cryogenic and room-temperatures is necessary in order not to miss structural details relevant for biological interpretations.

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