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Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10603095/ https://www.ncbi.nlm.nih.gov/pubmed/37884503 http://dx.doi.org/10.1038/s41467-023-42544-4 |
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author | Rzechorzek, Neil J. Kunzelmann, Simone Purkiss, Andrew G. Silva Dos Santos, Mariana MacRae, James I. Taylor, Ian A. Fugger, Kasper West, Stephen C. |
author_facet | Rzechorzek, Neil J. Kunzelmann, Simone Purkiss, Andrew G. Silva Dos Santos, Mariana MacRae, James I. Taylor, Ian A. Fugger, Kasper West, Stephen C. |
author_sort | Rzechorzek, Neil J. |
collection | PubMed |
description | Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. |
format | Online Article Text |
id | pubmed-10603095 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-106030952023-10-28 Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 Rzechorzek, Neil J. Kunzelmann, Simone Purkiss, Andrew G. Silva Dos Santos, Mariana MacRae, James I. Taylor, Ian A. Fugger, Kasper West, Stephen C. Nat Commun Article Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. Nature Publishing Group UK 2023-10-26 /pmc/articles/PMC10603095/ /pubmed/37884503 http://dx.doi.org/10.1038/s41467-023-42544-4 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Rzechorzek, Neil J. Kunzelmann, Simone Purkiss, Andrew G. Silva Dos Santos, Mariana MacRae, James I. Taylor, Ian A. Fugger, Kasper West, Stephen C. Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title | Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title_full | Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title_fullStr | Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title_full_unstemmed | Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title_short | Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 |
title_sort | mechanism of substrate hydrolysis by the human nucleotide pool sanitiser dnph1 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10603095/ https://www.ncbi.nlm.nih.gov/pubmed/37884503 http://dx.doi.org/10.1038/s41467-023-42544-4 |
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