Bacteria and eukaryotes post-transcriptionally modify tRNAs to modulate their activity. The 7-deazaguanosine derivative queuosine is a critical modification that is required for both translation accuracy and efficiency for the tRNAs that decode His, Tyr, Asp, Asn codons. Although bacteria can both synthesize queuosine de novo and salvage precursors from the environment, eukaryotes are required to salvage the queuine base to make the final modification in their tRNAs. It has been suggested that queuine could be a longevity vitamin and an important micronutrient for human health in recent studies. A gene that is presumed to encode a queuosine dedicated nucleoside hydrolase in bacterial queuine salvage pathways of many entero-pathogens (e.g., Clostridioides difficile) has been identified through bioinformatics. The physiological relevance of nucleoside hydrolases of these pathogens remains elusive and remarkably, neither nucleoside hydrolase activity nor the encoding genes have ever been detected in mammals making this nucleoside hydrolase a potential orthogonal therapeutic target. We optimized heterologous protein overexpression conditions and purified enzyme to greater homogeneity using size exclusion chromatography to further study the structure and function for this enzyme using X-ray crystallography, kinetics, and mutagenesis. Native PAGE showed that the enzyme exists as a homodimer. Multiple commercial crystallization screens were used in sitting drop vapor diffusion experiments to crystallize purified nucleoside hydrolase. Protein crystal hits obtained from these screens were further optimized using the hanging drop vapor diffusion to obtain crystals that diffracted to a resolution of 3.6 Å. To gain further structural insights into substrate binding and enzyme specificity, the protein was co-crystallized with D-ribose and queuine. We have identified the residues involved in the coordination of the 7-deazaguanine and cyclopentenediol ring of queuine moiety which are different from the typically conserved residues known to be involved in the coordination of purine base moieties. Structural insights gained will be used to guide the kinetics and mutagenesis experiments to functionally characterize the enzyme. The structural and functional information gained from this study will be invaluable to design small molecules to prevent queuosine salvage of these bacterial pathogens thus developing targeted antibiotics to prevent the diseases they cause.

Structural insights into nucleoside hydrolase involved in queuine salvage of gut pathogen Clostridioides difficile

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