Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis

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, MbNHase(Delta 238-257) and MbNHase(Delta 219-272), were prepared in which the (His)(17) sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an alpha(2)beta(2) 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 similar to 55% as active as the WT enzyme when expressed in the absence of the Co-type activator (epsilon) protein from Pseudonocardia thermophila JCM 3095 (PtNHase(ac)(t)) but 28% more active when expressed in the presence of PtNHase(act). MbNHase(Delta 219-272) exhibits similar to 55% and similar to 89% of WT activity, respectively, when expressed in the absence or presence of PtNHase(ac)(t). Proteolytic cleavage of MbNHase provides an alpha(2)beta(2) heterotetramer that is modestly more active compared to WT MbNHase (k(cat) = 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.