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.

Biosynthesis of the Urease Metallocenter

“…UreG: GTPase for Urease Activation-In contrast to UreD/ UreH and K. aerogenes UreF, which are insoluble, UreG is soluble and has been characterized from several sources (43)(44)(45)(46)(47) (46). X-ray absorption spectroscopy of the zincbound protein revealed a trigonal bipyramidal site including two His and two Cys residues, likely positioned at the subunit interface (49).…”

“…X-ray absorption spectroscopy of the zincbound protein revealed a trigonal bipyramidal site including two His and two Cys residues, likely positioned at the subunit interface (49). The soybean (Glycine max) protein also exhibits a monomer/dimer equilibrium, with the dimer stabilized by Zn 2ϩ , but the binding thermodynamics are quite complex (47). No crystal structure is available for free UreG, perhaps related to its intrinsic disorder (50); however, UreG homology models (44 -47) have been created by using HypB (required for nickel insertion into [Ni-Fe] hydrogenase) (51, 52) from Methanocaldococcus jannaschii (a dimeric GTPase with a dinuclear zinc site at the subunit interface (53)) as the template, and the structure of the H. pylori UreH:UreF:UreG complex is known (Fig.…”

“…3 The latter complex has two protomers of each peptide, with the two UreG molecules in contact as expected for a protein able to dimerize. Although UreG is a GTPase, the free protein exhibits slow (44,45,47) or no (43,46,48) GTPase activity. When present in urease activation complexes, GTPase activity is observed (54), and substitution of a key residue in the GTPbinding P-loop motif of K. aerogenes or H. pylori UreG abolishes the cell's ability to make active urease (43,55).…”

Nickel and Human Health

“…The nickel storage role is normally carried out by a His-rich pattern: this motif may constitute a whole protein, such as in the case of Hpn or HspA, two nickel accumulators found in Helicobacter pylori [ 123 -125 ], or may be located at the C-or at the N-terminus of a specifi c chaperone, as in the case of several UreE [ 126 ] and of UreG from Mycobacterium tuberculosis [ 127 ], Streptomyces coelicolor and Glycine max [ 128 ] for urease, HypB from Bradyrhizobium japonicum [ 129 ] and SlyD from Escherichia coli [ 130 ] for hydrogenase, and CooJ from Rhodospirillum rubrum [ 131 ] for CO dehydrogenase.…”

Unfoldome variation upon plant-pathogen interactions: strawberry infection by Colletotrichum acutatum

“…This characteristic allows IDPs to act as central hubs during multiple signal perception and transduction Tompa et al 2005), as observed also in plants (Kragelund et al 2012). Therefore, even though protein flexibility was found in some enzymes, possibly regulating their catalytic activity (Kathiriya et al 2014;Real-Guerra et al 2012;Schulenburg and Hilvert 2013;Zambelli et al 2012), IDPs mainly show Abstract Intrinsically disordered proteins (IDPs) are proteins that lack secondary and/or tertiary structure under physiological conditions. These proteins are very abundant in eukaryotic proteomes and play crucial roles in all molecular mechanisms underlying the response to environmental challenges.…”