Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity

Abstract

The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif (i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1.

Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana

“…Also, the flexibility of short loop regions might be a structural basis for recognizing distinct metabolites and has proved to be essential for discriminating structurally similar substrates [47]. Moreover, behind the broad substrate specificity of the Human Nudix Hydrolase MTH1, is the substrate-dependent shift in the protonation state of neighboring aspartic residues (Asp119 and Asp120) buried in the ligand-binding site [48], as they are Asp28 and Asp30 at AtSgpp motif 1. On this basis, the AtSgpp cap domain could be presumed as the molecular regulator that pivots the active site loop-5 motion to the particular orientation that would occupy diverse phosphosugar substrates, allowing different hydrogen-bonding pattern in the core domain upon cap closure.…”

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

“…(A) E. coli MutT recognizing oxodGMP in the syn conformation (3A6T) [158]. (B) human MTH1 recognizing oxodGTP in the anti conformation (5GHI) [163]. Numbers indicate the distances between putative hydrogen bond donors and acceptors; in (B), O 6 of oxoG is suggested to be protonated, and either O 6 or N1 can form a hydrogen bond at any given moment.…”

“…Also, Phe35 is conserved in MutT but replaced with Gly in MTH1 sequences. The structures of MTH1 from several vertebrate species, including humans, bound to a variety of modified nucleotides, revealed a binding site quite similar to MutT but a surprisingly different mode of oxoG recognition [163,[165][166][167][168]. In the active site pocket, the sugar of oxodGTP is flipped~180 • relative to the MutT structure, and the damaged base is anti, coplanar with oxoG base in MutT but orthogonal to it in this plane.…”

Crystal Structure and Substrate Specificity of the 8-oxo-dGTP Hydrolase NUDT1 from Arabidopsis thaliana

“…There is one 8-oxo-dGTP ligand and two magnesium ions per substrate binding site, the latter of which exhibits octahedral coordination to the ligand, several water molecules, and the Nudix motif ( Figure 1B). The structure of apo 7,13,42,43 Based on this observation we made single and double mutants at these positions to assess the role Asn76 and Ser89 play in the recognition of oxidized and non-oxidized nucleotides by AtNUDT1.…”