Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 μM. It exhibited mixed inhibition, with the Ki value being 26.28 μM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and β-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.