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.

Structure-Guided Insight into the Specificity and Mechanism of a Parasitic Nucleoside Hydrolase

“…Two examples of this include His241 of Crithidia fasciculata IUNH and Asp40 of Trypanosoma vivax IAGNH. , None of these residues are conserved in AGNH in the aligned primary structure (Figure S2), nor are they present in the homologous locations of the tertiary active-site structure, suggesting that AGNH may impart specificity and operate through a different mechanism than any of these purine or pyrimidine NHs. A recent review by Degano suggested groupings of the characterized NH enzymes into distinct structural classes . The overall fold of AGNH matches that of Degano’s Structural Group I, but the active site lacks the histidine leaving-group activator common to Group I enzymes .…”

“…Additionally, 64 sequences in the alignment were observed to have a cysteine in the position analogous to Ala238, suggesting that these enzymes may represent additional NH enzymes belonging to Degano’s Structural Group III . It was noted that all but two of these sequences were from eukaryotic organisms, including the two which have been structurally characterized (PDB 1MAS and 5MJ7).…”

“…A recent review by Degano suggested groupings of the characterized NH enzymes into distinct structural classes . The overall fold of AGNH matches that of Degano’s Structural Group I, but the active site lacks the histidine leaving-group activator common to Group I enzymes . AGNH therefore represents another example of NH enzymes with noncanonical catalytic residues.…”

“…AGNH therefore represents another example of NH enzymes with noncanonical catalytic residues. Other NH enzymes containing a noncanonical active-site cysteine residue were classified by Degano as Structural Group III; however, AGNH does not fall within this group as the cysteine position is occupied by alanine. − …”

Bifunctional NadC Homologue PyrZ Catalyzes Nicotinic Acid Formation in Pyridomycin Biosynthesis

“…From the biochemical point of view, the ribose hydrolysis of NAR is similar to the cleavage of the N-glycosidic bond of ribonucleosides catalyzed by nucleoside hydrolases. Usually, the ribose hydrolases need a general basic residue (aspartate or glutamate) to abstract a proton from a water molecule, and the water molecule then performs the nucleophilic attack on the glycosidic bond. − Based on the molecular docking model, the negatively charged residues of D185, E213, and D234 close to the NAMN ribose group were investigated by site-directed mutagenesis. In vitro biochemical reaction results showed that these mutant proteins D185A, E213A, D234A, and D296A did not affect the ribose hydrolysis activity, indicating that none of the above four acidic amino acids could act as a basic residue for the ribose hydrolysis of NAR (Figure S16).…”