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

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Abstract

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.

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Abbreviations

A:

Adenosine

AAG:

Alkyladenine DNA glycosylase

C:

Cytidine

CU–NH:

Cytidine–uridine nucleoside hydrolase

G:

Guanosine

I:

Inosine

IAG–NH:

Inosine–adenosine–guanosine nucleoside hydrolase

IG–NH:

Inosine–guanosine nucleoside hydrolase

IU–NH:

Inosine–uridine nucleoside hydrolase

MCPB:

Metal center parameter builder

MD:

Molecular dynamics

NH:

Nucleoside hydrolase

pAPIR:

p-Aminophenyliminoribitol

PDB:

Protein data bank

RESP:

Restrained electrostatic potential

rms:

Root-mean-square

rmsd:

Root-mean-square deviation

U:

Uridine

UDG:

Uracil DNA glycosylase

X:

Xanthosine

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Acknowledgements

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.

Funding

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.

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  1. Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, AB, T1K 3M4, Canada

    Stefan A. P. Lenz & Stacey D. Wetmore

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Correspondence to Stacey D. Wetmore.

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Electronic supplementary material

Supporting information—Discussion of wild-type (Asp15 His239) and single mutant (Asp15– His239+) data, active site parameters (Tables S1 and S2), replicate backbone rmsd (Table S3), key distances (Table S4), hydrogen-bonding (Tables S5 and S8), active site torsional angles (Tables S6 and S9), MM-GBSA binding energies (Table S7); structure and numbering of calcium-ligating residues (Figure S1), overlay of MD representative structure and X-ray crystal structure for wild-type CU–NH-I (Figure S2), radial distribution plot of water density with respect to Ca2+, stacked histogram of pseudorotational angle occupancy (Figure S4), overlays of MD representative structures and water distribution figures (Figure S5–S9). Full citation for references 6, 20, 22, 26, 48, and 51. Below is the link to the electronic supplementary material.

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Lenz, S.A.P., Wetmore, S.D. Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations. J Comput Aided Mol Des 32, 1375–1388 (2018). https://doi.org/10.1007/s10822-018-0178-y

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