Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

Abstract

Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N-glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine-specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine-specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site-directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens

“…S6). However, the typical conserved residues known to be involved in the coordination of the pyrimidine or purine moieties in the characterized enzymes among the family are not present (45). As queuosine is a 7-deazaguanine carrying a cyclopentenediol ring linked at position 7 through an aminomethyl linkage, a more sizeable substrate pocket to accommodate the queuine moiety is required.…”

Enzymatic Transition States and Drug Design

“…Others, for example the protozoan GI-nucleoside hydrolase, strongly prefer 6-oxypurine leaving groups, and the IAG-nucleoside hydrolase from T. brucei prefers these purine leaving groups. In contrast, AMP nucleosidase is specific for adenine, and purine nucleoside phosphorylase is specific for 6-oxypurine leaving groups …”

“…In contrast, AMP nucleosidase is specific for adenine, and purine nucleoside phosphorylase is specific for 6-oxypurine leaving groups. 85…”

Structure, Oligomerization and Activity Modulation in N-Ribohydrolases

“…Based on the nature of the active-site residues that are present in the β3-α3 loop (His vs. Trp in group I and II NHs) and before the last Asp in the Ca 2+ coordination sphere (again a His in group I vs. a Trp in group II), a third structural group was anticipated. Indeed, a group of NH proteins from metazoa including C. elegans and X. laevis display a Cys residue rather than Trp260 or His241 [ 33 , 71 ]. Structural characterization of the C. elegans NH showed that it shares the highest catalytic similarity to group II NHs, but displays an overall structure that is more similar to group I NHs, including its tetrameric quaternary structure [ 71 ].…”

“…Indeed, a group of NH proteins from metazoa including C. elegans and X. laevis display a Cys residue rather than Trp260 or His241 [ 33 , 71 ]. Structural characterization of the C. elegans NH showed that it shares the highest catalytic similarity to group II NHs, but displays an overall structure that is more similar to group I NHs, including its tetrameric quaternary structure [ 71 ]. Site-directed mutagenesis demonstrated that the Cys residue is involved in the leaving-group activation, albeit through a yet-unidentified mechanism.…”