Structure, Oligomerization and Activity Modulation in N-Ribohydrolases

Abstract

Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides (N-ribohydrolases, NHs) with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases.

Keywords: N-ribohydrolases; drug design; quaternary structure; ribosides; structural enzymology.

Figure 1

N-ribosidic bond cleavage in biological systems. Phosphorolysis catalyzed by NPs or hydrolysis by NHs of ribosides (here, purine riboside is shown as a general example) releases the nitrogenous base, and either ribose 1-phosphate or ribose is formed.

Figure 2

NH fingerprint sequence in NH enzymes and NH-like proteins. In bold are the Asp residues that are involved in the coordination of the Ca²⁺ ion. Underlined are the amino acid residues in each group that have been demonstrated through site-directed mutagenesis to be involved in catalysis. The color scheme matches the one used in the active site representations. The sequences are taken from the deposited PDB files (PDB codes 2MAS, 4I71, 5MJ7, 4P5F).

Figure 3

Group I NH structure. (A) Overall topology diagram of group I NHs. The major secondary structural elements shared by all group I NHs so far characterized are indicated (α-helices as cylinders, β-strands as arrows). Occasionally, additional short helical segments are identified through automated analysis, thus the numbering may differ. Strands β8 and β9 (green) are components of the major interaction surface between group I monomers for tetramer assembly, while elements colored in cyan compose the minor interaction surface. (B) Ribbon diagram of the monomer of the epitomic C. fasciculata IU-NH (PDB code 2MAS). (C) Active site architecture of group I NHs. Here, the complex between the E. coli RihB/YeiK enzyme and inosine is shown. The Ca²⁺ ion octacoordination is composed of three Asp residues from the NH sequon, the carbonyl oxygen of a non-conserved amino acid (commonly Thr, Val or Ile), one water molecule, and the O2’ and O3’ ribosyl hydroxyls. In the absence of a substrate, two ordered waters occupy the hydroxyl positions. Hydrogen bonds between the enzyme, substrate, and catalytic water are colored blue. Amino acids are colored according to the scheme in Figure 2. (D) Conformational changes in group I NHs on binding of active site ligands. The α3-β3 loop and helix α8 are highly flexible in the structure determined in the absence of a ligand (gray tube), and undergo a transition to a defined conformation when active site ligands are bound (green tube, with the segments undergoing a structural transition colored orange).

Figure 4

Group II NH structure. (A) Topology diagram of group II NHs. Four helical segments (α3.1, α3.1, α7, and α8) are part of the monomer-monomer interface. (B) Ribbon diagram of the monomer of the T. brucei brucei IAG-NH (PDB code 4I71). (C) Active site of group II NHs. The T. brucei brucei IAG-NH in complex with the UAMC-0063 inhibitor shows the aromatic stacking between the aglycone and two signature Trp residues, along with specific hydrogen bonding to the base substituents. (D) Flexible loop conformational selection. The flexible loop in unliganded IAG-NH spanning residues 245–255 becomes ordered (pink) on binding of substrates or competitive inhibitors.

Figure 5

Oligomerization of NH proteins. (A) Ribbon diagram of the tetrameric group I IU-NH from C. fasciculata. The quaternary structure is achieved through two interaction surfaces, a major (colored green) one largely involving the β8 and β9 strands that are not part of the central core sheet, and a minor (cyan) one that is composed of residues from the loop connecting strand β3 to helix α3, and helices α4.1 and α5.2. (B) Ribbon diagram of the dimeric plant NH. This dimer, oriented in the figure as the C. fasciculata isozyme in the top panel, is stabilized by extensive interactions between residues corresponding to the major surface in the group I tetramers. The conformation of the secondary structural elements corresponding to the minor interface is conserved; thus, the different amino acid compositions at the β3-α3 junction and α4.1 and α5.2 helices prevent the formation of the tetrameric structure. (C) Dimeric group II NH. Deletions in the β8-β9 region and the presence of two additional helices α3.1 and α3.2 in the region joining strand β3 to β4 allow a different mode of intermolecular stabilization.

Figure 6

Structural comparison between NH proteins. Pairwise superposition of the T. brucei brucei IAG-NH (red), C. elegans NH (cyan), and X. oryzae XopQ onto the C. fasciculata IU-NH (yellow) highlights the conservation of the core b-sheet, while the surrounding secondary structural elements assume diverse orientations, affecting their activity and oligomerization.

Figure 7

Iminoribitol-based NH inhibitors. (A) Substituted phenyliminoribitols are effective, transition state-like inhibitors of the Crithidia IU-NH. (B) 4-nitrophenyl riboamidrazone, a nanomolar inhibitor of the C. fasciculata isozyme. (C) Immucillins, originally identified as transition state-like NP inhibitors, are also effective inhibitors of the group II trypanosomal IAG-NHs and IG-NH. (D) Methylaryl iminoribitols show high affinity toward trypanosomal IAG-NHs with high selectivity, being much weaker NP inhibitors.

Figure 8

Prodrug activation by NHs. (A) IAG-NHs efficiently convert the prodrug 6-methylpurine riboside to 6-methylpurine, whose incorporation in RNA affects protein synthesis. (B) Non-specific IU-NHs or pyrimidine-selective CU-NHs hydrolyze the N-glycosidic bond in 5-fluorouridine, leading to release of 5-fluorouracil. The fluorinated pyrimidine is incorporated in nucleotides that hinder the RNA secondary structure, and the deoxy mononucleotide inhibits the key enzyme thymidylate synthase.