The iron-type nitrile hydratase activator protein is a GTPase.
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 GTP to GDP with a kcatvalue of 1.2 × 10-3s-1and a Kmvalue of 40 μM in the presence of 5 mM MgCl2in 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 Kdof 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.
Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Rd, Chicago, IL 60660, U.S.A.
Natalie
N
Gumataotao
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201, U.S.A.
K P Wasantha
KP
Lankathilaka
Department of Physics, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, U.S.A.
Brian
B
Bennett
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201, U.S.A. richard.holz@marquette.edu.
Richard C
RC
Holz
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016929:Rhodococcus equi / Q000737:chemistry* / Q000201:enzymology; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D007501:Iron; D020558:GTP Phosphohydrolases; C481966:YjiA protein, E coli; D006836:Hydro-Lyases; C048831:nitrile hydratase
enzyme kinetics; metal transport; nitrile hydratase; protein biosynthesis