8Y1Q
Crystal structure of Aquifex aeolicus dUTPase complexed with dUMP.
Summary for 8Y1Q
| Entry DOI | 10.2210/pdb8y1q/pdb |
| Descriptor | Deoxyuridine 5'-triphosphate nucleotidohydrolase, 2'-DEOXYURIDINE-5'-MONOPHOSPHATE, MAGNESIUM ION, ... (4 entities in total) |
| Functional Keywords | dutpase, dutp, dump, hyperthermophile, aquifex aeolicus, hydrolase |
| Biological source | Aquifex aeolicus |
| Total number of polymer chains | 3 |
| Total formula weight | 50182.10 |
| Authors | |
| Primary citation | Fukui, K.,Kondo, N.,Murakawa, T.,Baba, S.,Kumasaka, T.,Yano, T. dUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application. Protein Sci., 33:e5185-e5185, 2024 Cited by PubMed Abstract: Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity. PubMed: 39440877DOI: 10.1002/pro.5185 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.42 Å) |
Structure validation
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