Journal of Computer-Aided Molecular Design

, Volume 32, Issue 12, pp 1375–1388 | Cite as

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations

  • Stefan A. P. Lenz
  • Stacey D. WetmoreEmail author


Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine–uridine nucleoside hydrolase (CU–NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU–NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU–NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU–NH are identified. Regardless of the wild-type or mutant CU–NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU–NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU–NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Graphical Abstract


Molecular dynamics Cytidine-uridine nucleoside hydrolase Inosine-uridine nucleoside hydrolase Substrate binding Hydrolysis of inosine 





Alkyladenine DNA glycosylase




Cytidine–uridine nucleoside hydrolase






Inosine–adenosine–guanosine nucleoside hydrolase


Inosine–guanosine nucleoside hydrolase


Inosine–uridine nucleoside hydrolase


Metal center parameter builder


Molecular dynamics


Nucleoside hydrolase




Protein data bank


Restrained electrostatic potential




Root-mean-square deviation




Uracil DNA glycosylase





Computational resources from the New Upscale Cluster for Lethbridge to Enable Innovative Chemistry (NUCLEIC) and those provided by Westgrid and Compute/Calcul Canada are greatly appreciated.


Support for this research was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC, Grant No. 2016–04568), the Canada Foundation for Innovation (Grant No. 22770) and the Board of Governors Research Chair Program at the University of Lethbridge. S.A.P.L. acknowledges NSERC (CGS-D), Alberta Innovates-Technology Futures (AI-TF) and the University of Lethbridge for student scholarships.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

10822_2018_178_MOESM1_ESM.docx (19.4 mb)
Supplementary material 1 (DOCX 19888 KB)


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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada

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