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

S. Waz, T. Nakamura, K. Hirata, Y. K. Ogawa, M. Chirifu, T. Arimori, T. Tamada, S. Ikemizu, Yusaku Nakabeppu, Y. Yamagata

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Abstract

The human MutT homolog 1 (hMTH1, hNUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anti-cancer 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 Asp120 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 of the relation 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 (Asp119 and Asp120) 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 anti-cancer drug development strategies targeting hMTH1.
Original languageEnglish
Pages (from-to)2785-2794
Number of pages10
JournalJournal of Biological Chemistry
Volume292
Issue number7
DOIs
Publication statusPublished - Feb 17 2017

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Substrate Specificity
Nucleotides
Kinetics
Substrates
Neoplasms
Hydrolases
Enzymes
Hydrogen Bonding
Nucleosides
Aspartic Acid
Crystal structure
Hydrolysis
Cell Death
X-Rays
nudix hydrolases
Escherichia coli
Protonation
Mutation
Bond length
Pharmaceutical Preparations

Cite this

Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity. / Waz, S.; Nakamura, T.; Hirata, K.; Ogawa, Y. K.; Chirifu, M.; Arimori, T.; Tamada, T.; Ikemizu, S.; Nakabeppu, Yusaku; Yamagata, Y.

In: Journal of Biological Chemistry, Vol. 292, No. 7, 17.02.2017, p. 2785-2794.

Research output: Contribution to journalArticle

Waz, S, Nakamura, T, Hirata, K, Ogawa, YK, Chirifu, M, Arimori, T, Tamada, T, Ikemizu, S, Nakabeppu, Y & Yamagata, Y 2017, 'Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity', Journal of Biological Chemistry, vol. 292, no. 7, pp. 2785-2794. https://doi.org/10.1074/jbc.M116.749713
Waz, S. ; Nakamura, T. ; Hirata, K. ; Ogawa, Y. K. ; Chirifu, M. ; Arimori, T. ; Tamada, T. ; Ikemizu, S. ; Nakabeppu, Yusaku ; Yamagata, Y. / Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 7. pp. 2785-2794.
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AU - Waz, S.

AU - Nakamura, T.

AU - Hirata, K.

AU - Ogawa, Y. K.

AU - Chirifu, M.

AU - Arimori, T.

AU - Tamada, T.

AU - Ikemizu, S.

AU - Nakabeppu, Yusaku

AU - Yamagata, Y.

PY - 2017/2/17

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N2 - The human MutT homolog 1 (hMTH1, hNUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anti-cancer 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 Asp120 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 of the relation 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 (Asp119 and Asp120) 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 anti-cancer drug development strategies targeting hMTH1.

AB - The human MutT homolog 1 (hMTH1, hNUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anti-cancer 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 Asp120 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 of the relation 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 (Asp119 and Asp120) 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 anti-cancer drug development strategies targeting hMTH1.

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JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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ER -