Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10156658 | Archives of Biochemistry and Biophysics | 2018 | 32 Pages |
Abstract
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, MbNHaseÎ238â257 and MbNHaseÎ219â272, 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 â¼55% 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 â¼28% more active when expressed in the presence of PtNHaseact. MbNHaseÎ219â272 exhibits â¼55% and â¼89% 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 (kcatâ¯=â¯163â¯Â±â¯4 vs 131â¯Â±â¯3 sâ1). 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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Authors
Xinhang Yang, Brian Bennett, Richard C. Holz,