Cobalt metallochaperone-mediated maturation of nitrile hydratases: Structural and functional insights

The Co-type nitrile hydratase activator (ɛ) protein from Pseudonocardia thermophila JCM 3095 (PtCo-type ε protein) was successfully expressed and purified, enabling the first detailed biochemical and spectroscopic characterization of Co(II) binding. The PtCo-type ε protein can hydrolyze GTP and ATP with k_cat values of 1.2 × 10^-3 and 0.6 × 10^-3 s^-1, respectively, in the presence of 5 mM MgCl₂ at pH 7.5. Removal of Co(II) decreased GTP hydrolysis rate by ∼40%, suggesting that cobalt binding may contribute to the proposed metallochaperone function of the PtCo-type ε protein. CD data revealed that the PtCo-type ε protein is ∼80% α-helical and ∼ 20% random coil and its secondary structure is not perturbed by nucleotide binding. UV-Vis, EPR and X-ray absorption spectroscopy show that the PtCo-type ε protein binds a single Co(II) ion in a five-coordinate O/N ligand environment, with EXAFS consistent with coordination by two histidine residues. As no X-ray crystal structure exists for any NHase ε protein, an AlphaFold3 model was developed that reveals five α-helices, consistent with CD data, and revealed a potential Co(II) binding site made up of H129, H132, and E134. A partial Walker B-like segment (99-104: PEALDE), together with a conserved 61-63: EWK sequence, may contribute to the proposed GTP/ATP binding site. Together, these results provide insight into the Co(II) binding and nucleotide hydrolysis by a Co-type NHase ε protein and establish a structural framework for understanding its role in NHase metallocentre assembly.