Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1
Abstract 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 molec...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-10-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-42544-4 |
| _version_ | 1797647264208912384 |
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| author | Neil J. Rzechorzek Simone Kunzelmann Andrew G. Purkiss Mariana Silva Dos Santos James I. MacRae Ian A. Taylor Kasper Fugger Stephen C. West |
| author_facet | Neil J. Rzechorzek Simone Kunzelmann Andrew G. Purkiss Mariana Silva Dos Santos James I. MacRae Ian A. Taylor Kasper Fugger Stephen C. West |
| author_sort | Neil J. Rzechorzek |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-11T15:13:48Z |
| format | Article |
| id | doaj.art-6e4d10f136754e82b50ca61ead5af62a |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-11T15:13:48Z |
| publishDate | 2023-10-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-6e4d10f136754e82b50ca61ead5af62a2023-10-29T12:29:35ZengNature PortfolioNature Communications2041-17232023-10-0114111110.1038/s41467-023-42544-4Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1Neil J. Rzechorzek0Simone Kunzelmann1Andrew G. Purkiss2Mariana Silva Dos Santos3James I. MacRae4Ian A. Taylor5Kasper Fugger6Stephen C. West7DNA Recombination and Repair Laboratory, The Francis Crick InstituteStructural Biology Science Technology Platform, The Francis Crick InstituteStructural Biology Science Technology Platform, The Francis Crick InstituteMetabolomics Science Technology Platform, The Francis Crick InstituteMetabolomics Science Technology Platform, The Francis Crick InstituteMacromolecular Structure Laboratory, The Francis Crick InstituteDNA Recombination and Repair Laboratory, The Francis Crick InstituteDNA Recombination and Repair Laboratory, The Francis Crick InstituteAbstract 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.https://doi.org/10.1038/s41467-023-42544-4 |
| spellingShingle | Neil J. Rzechorzek Simone Kunzelmann Andrew G. Purkiss Mariana Silva Dos Santos James I. MacRae Ian A. Taylor Kasper Fugger Stephen C. West Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1 Nature Communications |
| 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 |
| url | https://doi.org/10.1038/s41467-023-42544-4 |
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