title: Crystal structures of the novel cytosolic 5'-nucleotidase IIIB explain its preference for m7GMP.
abstract: 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.
authors.affiliation: Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Monecke
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Buschmann
authors.affiliation: Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Piotr
authors.initials: P
authors.lastname: Neumann
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Wahle
authors.affiliation: Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Göttingen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
mesh_terms: D015720:5'-Nucleotidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D003562:Cytidine / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012314:RNA Cap Analogs / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D007527:Isoenzymes; D012314:RNA Cap Analogs; D011994:Recombinant Proteins; C011925:7-methylguanosine-5'-monophosphate; D003562:Cytidine; D006147:Guanine; C008450:7-methylguanine; D015720:5'-Nucleotidase