title: Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein.
abstract: 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 Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 ± 0.01 μM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) 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.
authors.affiliation: Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, Porto Alegre, RS 91501-970, Brazil. rafael.guerra@ufrgs.br
authors.forename: Rafael
authors.initials: R
authors.lastname: Real-Guerra
authors.forename: Fernanda
authors.initials: F
authors.lastname: Staniscuaski
authors.forename: Barbara
authors.initials: B
authors.lastname: Zambelli
authors.forename: Francesco
authors.initials: F
authors.lastname: Musiani
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
authors.forename: Célia R
authors.initials: CR
authors.lastname: Carlini
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D020558:GTP Phosphohydrolases; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030262:Soybean Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008670:Metals; D011994:Recombinant Proteins; D030262:Soybean Proteins; D009532:Nickel; D020558:GTP Phosphohydrolases; C406502:ureG protein, Glycine max; D015032:Zinc