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

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, MbNHase?238-257 and MbNHase?219-272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an ?2?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 ?55% as active as the WT enzyme when expressed in the absence of the Co-type activator (?) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ?28% more active when expressed in the presence of PtNHaseact. MbNHase?219-272 exhibits ?55% and ?89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an ?2?2 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.

SUBMITTER: Yang X

PROVIDER: S-EPMC6201762 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Yang Xinhang X Bennett Brian B Holz Richard C RC

Archives of biochemistry and biophysics 20180908

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 an ...[more]

PMID: 30205086

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Project description:BackgroundNitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.Methodology/principal findingsHere we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.Conclusions/significanceThe evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.

Dataset Information

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

Publications

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

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