Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni2+ or Zn2+, secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni2+ and Zn2+ was determined by isothermal titration calorimetry. rGmUreG binds Zn2+ or Ni2+ differently, presenting a very tight binding site for Zn2+ (K d = 0.02 ± 0.01 μM) but not for Ni2+, thus suggesting that Zn2+ may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn2+ stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated.
Plant Molecular Biology – Springer Journals
Published: Jan 22, 2012
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