Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis
2018
Yang, Xinhang | Bennett, Brian | Holz, Richard C.
The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ²³⁸⁻²⁵⁷ and MbNHaseΔ²¹⁹⁻²⁷², were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2β2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseᵃᶜᵗ) but ∼28% more active when expressed in the presence of PtNHaseᵃᶜᵗ. MbNHaseΔ²¹⁹⁻²⁷² exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseᵃᶜᵗ. Proteolytic cleavage of MbNHase provides an α2β2 heterotetramer that is modestly more active compared to WT MbNHase (kcat = 163 ± 4 vs 131 ± 3 s⁻¹). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.
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