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

• Published in: Archives of biochemistry and biophysics 2018-11, Vol.657, p.1-7
• Main Authors: Yang, Xinhang, Bennett, Brian, Holz, Richard C.
• Format: Article
• Language: English
• Subjects: And metal insertion; Cobalt; Enzyme kinetics; Metal transport; Nitrile hydratase; Protein biosynthesis
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.08.013

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. •Metallocentre assembly in the eukaryotic nitrile hydratase from Monosiga brevicollis (MbNHase).•WT MbNHase expresses as a single polypeptide with fused α- and β-subunits linked by a (His)17 and an insert region.•Two insert region mutant MbNHase enzymes were examined, MbNHaseΔ238−257 and MbNHaseΔ219−272.•Neither the (His)17 nor the entire insert region are required for metallocentre assembly and maturation.