Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP.
5'-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of disea...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3946280?pdf=render |
| _version_ | 1818310196524482560 |
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| author | Thomas Monecke Juliane Buschmann Piotr Neumann Elmar Wahle Ralf Ficner |
| author_facet | Thomas Monecke Juliane Buschmann Piotr Neumann Elmar Wahle Ralf Ficner |
| author_sort | Thomas Monecke |
| collection | DOAJ |
| description | 5'-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5'-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5'-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine- and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through π-π interactions and distinguished from unmodified guanosine by additional cation-π coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 5'-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m7GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m7GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions. |
| first_indexed | 2024-12-13T07:42:13Z |
| format | Article |
| id | doaj.art-6c30d2058f384b598ac7d067178c28fe |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T07:42:13Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-6c30d2058f384b598ac7d067178c28fe2022-12-21T23:54:57ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0193e9091510.1371/journal.pone.0090915Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP.Thomas MoneckeJuliane BuschmannPiotr NeumannElmar WahleRalf Ficner5'-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5'-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5'-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine- and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through π-π interactions and distinguished from unmodified guanosine by additional cation-π coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 5'-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m7GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m7GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions.http://europepmc.org/articles/PMC3946280?pdf=render |
| spellingShingle | Thomas Monecke Juliane Buschmann Piotr Neumann Elmar Wahle Ralf Ficner Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. PLoS ONE |
| title | Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. |
| title_full | Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. |
| title_fullStr | Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. |
| title_full_unstemmed | Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. |
| title_short | Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP. |
| title_sort | crystal structures of the novel cytosolic 5 nucleotidase iiib explain its preference for m7gmp |
| url | http://europepmc.org/articles/PMC3946280?pdf=render |
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