The biosynthesis of [NiFe]-hydrogenase and urease in Helicobacter pylori requires several accessory proteins for proper assembly of the nickel-containing active sites. Critical to the maturation of both enzymes in H. pylori is the GTPase HypB. In this work, the metal-binding properties of H. pylori HypB (HpHypB) were investigated and a link between metal binding and the other biochemical properties of HpHypB was established. HpHypB binds stoichiometric nickel or zinc with nanomolar affinities, in partially overlapping sites located between two major GTPase motifs. Upon metal binding, the GTP hydrolysis activity and oligomeric properties of the protein are modulated. Furthermore, the stoichiometry and affinity of the nickel is altered when HpHypB is bound to nucleotide, a change not observed for zinc. Mutagenesis of the metal ligands suggest that a conserved cysteine is responsible for transducing the metal-bound state to altered GTPase activity and a conserved histidine is a required nickel ligand only in the nucleotide-bound state. Together, these results suggest that the metal-binding and GTP hydrolysis properties of HpHypB are intimately linked and may comprise a mechanism through which the [NiFe]-hydrogenase and urease maturation pathways can discriminate between Ni(II) and Zn(II). Characterization of the Escherichia coli GTPase YjiA, a member of the same GTPase family as HpHypB, demonstrated that YjiA can bind Ni(II), Zn(II), or Co(II) at a site in a similar location as in HpHypB. Metal binding also regulates the GTPase activity and oligomerization of YjiA. This finding suggests that metal-responsive GTPase activity may be a trait of this family of GTPases. Together, this work describes a unique link between the metal-binding and biochemical properties of the G3E GTPases and provides insight into the role of HpHypB in [NiFe]-hydrogenase and urease maturation.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/43738 |
| Date | 14 January 2014 |
| Creators | Sydor, Andrew |
| Contributors | Zamble, Deborah |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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