The iron-type nitrile hydratase activator protein is a GTPase

Abstract

Abstract:The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.Biochemical Journal, Vol 474, No. 2 (January 15, 2017): pg. 247-258. DOI. This article is © Portland Press Limited and permission has been granted for this version to appear in e-Publications@Marquette. Portland Press Limited does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Portland Press Limited. 2GTP to GDP with a kcat value of 1.2 × 10 −3 s −1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.

The Fe-type nitrile hydratase from Rhodococcus equi TG328-2 forms an alpha-activator protein complex

“…While (ε) proteins may indeed dictate the identity of the metal ion in Co-type NHases and participate in the controlled oxidation of the two equatorial Cys residues, no biochemical or structural data exist to support this hypothesis in Fe-type NHases. Recently, the Fe-type ReNHase (ε) protein was heterologously overexpressed and purified [16]. These data established, for the first time, that the ReNHase (ε) protein is a member of the COG0523 subfamily of G3E P-loop GTPases and that GTPase activity is regulated by metal binding.…”

“…The original plasmid had the NHase α, β, and activator genes in tandem. Therefore, separate pET-28a + plasmids containing only the His 6 -tagged ReNHase TG328-2 α-subunit and the His 6 -tagged ReNHase TG328-2 β-subunit genes were synthesized by Genscript, while the pMCSG9 plasmid containing the His 6 -MBP-ReNHase TG328-2 (ε) protein with a Tobacco Etch Virus (TEV) protease cleavage site between MBP and the ReNHase TG328-2 (ε) protein, as previously described, was used to express the ReNHase TG328-2 (ε) protein [16]. All plasmid sequences were confirmed using automated DNA sequencing at the Functional Biosciences DNA sequencing facility.…”

“…Recently, an Fe-type NHase (ε) protein was expressed and characterized revealing that it is a member of the COG0523 subfamily of G3E P-loop GTPases and that GTPase activity is regulated by metal binding [16,17]. Since the role of the Fe-type (ε) protein in NHase metallocenter assembly is directly related to NHase structure and function, investigating this process will identify aspects of metallocenter assembly that are essential for catalysis.…”

Metallochaperones and metalloregulation in bacteria

“…Each enzyme class, while structurally homologous, appears to employ very distinct activator or metallochaperone strategies that catalyse the assembly and oxidative maturation of the Fe III compared with Co III active sites. In the case of the Fe III NHases, a G3E family P-loop GTPase, Rhodococcus equi TG328-2, found in the same genomic locus, has been recently characterized and shown to exhibit weak GTPase activity [70]. This GTPase binds Co II to a CCIC (where C is cysteine) cysteine thiolate-rich motif and exhibits very high sequence similarity to A. baumannii ZigA, which we believe is representative of the COG0523 subfamily of P-loop GTPases that are up-regulated under conditions of extreme zinc limitation [15].…”

Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones

“…Overall, the Co II titration experiments provide evidence for two tetrahedral or pseudotetrahedral sites with distinct affinities and confirm that the high-affinity site contains at least one Cys ligand and that both sites bind Zn II preferentially over Co II . 40 Given the high sequence similarity of AbZigA and SaZigA with the Fe-type nitrile hydratase activator (Nha3) 41 (Figures 2A and S2) and the fact that ΔzigA Ab exacerbates both Zn II and Fe II starvation induced by CP, 13 we explored the possibility that these GTPases also bind Fe II . We used the same Zn II chelator, which, given its lower affinity for Fe II , would allow us to evaluate Fe II binding affinities in the 10 4 -10 6 M −1 affinity range.…”