Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis. Arch Biochem Biophys 2018 Nov 01;657:1-7
Date
09/12/2018Pubmed ID
30205086Pubmed Central ID
PMC6201762DOI
10.1016/j.abb.2018.08.013Scopus ID
2-s2.0-85053206138 (requires institutional sign-in at Scopus site) 2 CitationsAbstract
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.
Author List
Yang X, Bennett B, Holz RCAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceCatalytic Domain
Choanoflagellata
Cobalt
Hydro-Lyases
Kinetics
Metalloproteins
Mutation
Protein Processing, Post-Translational
Protein Subunits
Proteolysis
Sequence Alignment
