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.

Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside analogs

“…3). Previous structural studies of intracellular 5’ nucleotidase family members show the catalytic core is formed by two aspartates in Motif I and two aspartates in Motif III oriented near each other [25–29]. Our homology model for NT5C1A suggests a similar catalytic core composed of p.D211, p.D213, p.D328, and p.D329 (blue).…”

“…Our homology model for NT5C1A suggests a similar catalytic core composed of p.D211, p.D213, p.D328, and p.D329 (blue). Prior analyses also include a lysine at the N terminal end of Motif III (proposed to be p.K320 for NT5C1A) interacting with the bound substrate's phosphate group [2,25,29]. However, our structural model predicts p.K320 (light blue) to be remote from the catalytic core, whereas p.K314 (red), which is evolutionarily conserved, is predicted to be more proximal and is more likely to provide the protons needed to stabilize the NT5C1A dephosphorylation reaction (Fig.…”

“…The intracellular 5’ nucleotidases share three highly conserved functional motifs as identified by solved crystal structures and predicted by sequence analyses to date [2,25–29]. Residues in Motif I and III are involved in catalysis and residues in Motif S are involved in substrate recognition.…”

The complex enzymology of mRNA decapping: Enzymes of four classes cleave pyrophosphate bonds

“…The enzyme is specific for 5′‐5′‐linked dinucleotides or very small capped oligonucleotides (H. Liu, Rodgers, Jiao, & Kiledjian, ; Z. Wang & Kiledjian, ); long mRNAs are not targeted (L. S. Cohen et al, ; H. Liu et al, ; Z. Wang et al, ). In metazoa, the resulting product, m 7 GMP, is further degraded by the GMP‐specific nucleotidase III into 7‐methylguanosine and inorganic phosphate (Buschmann et al, ; Monecke, Buschmann, Neumann, Wahle, & Ficner, ); in yeast, m 7 GMP degradation yields a different product via an unidentified enzyme (Buschmann et al, ; van Dijk, Le Hir, & Séraphin, ). It has now become clear that DcpS has a second function—the degradation of m 7 GDP, originating either from the decapping reaction of the 5′→3′ decay pathway, for example, by Dcp2, or from the scavenger activities of the Fhit/Hnt2 enzymes (discussed below) (Taverniti & Séraphin, ); this had been a matter of debate in the past (H. Liu et al, ; Malys & McCarthy, ; van Dijk et al, ; Wypijewska et al, ).…”

Enzyme Promiscuity: Engine of Evolutionary Innovation

“…With differences in substrate specificity, localization, oligomerization state, and size, seven different 5Ј-nucleotidases have been characterized in humans. Among these, the HAD superfamily member cytosolic 5Ј-nucleotidase III-like protein (cN-IIIB) from humans and Drosophila has been recently described and has been shown to have broad activity against multiple substrates (35). This enzyme efficiently dephosphorylates CMP, UMP, GMP, AMP, and notably, the modified purine 7-methylguanosine monophosphate (m 7 GMP, Fig.…”