A Bifunctional dCTP Deaminase-dUTP Nucleotidohydrolase from the Hyperthermophilic Archaeon Methanocaldococcus jannaschii
(2003) In Journal of Biological Chemistry 278(23). p.20667-20672- Abstract
- By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the - and -phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of... (More)
- By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the - and -phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 µM). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 µM) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/124607
- author
- Björnberg, Olof LU ; Neuhard, Jan and Nyman, Per-Olof LU
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 278
- issue
- 23
- pages
- 20667 - 20672
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- wos:000183230500030
- scopus:17744410903
- ISSN
- 1083-351X
- DOI
- 10.1074/jbc.M213010200
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Biochemistry and Structural Biology (S) (000006142), Biology building (Closed 2011) (011008000)
- id
- 0744dd65-60d3-4949-a510-b366e2d1806f (old id 124607)
- date added to LUP
- 2016-04-01 11:58:33
- date last changed
- 2022-01-26 21:00:35
@article{0744dd65-60d3-4949-a510-b366e2d1806f, abstract = {{By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the - and -phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 µM). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 µM) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.}}, author = {{Björnberg, Olof and Neuhard, Jan and Nyman, Per-Olof}}, issn = {{1083-351X}}, language = {{eng}}, number = {{23}}, pages = {{20667--20672}}, publisher = {{American Society for Biochemistry and Molecular Biology}}, series = {{Journal of Biological Chemistry}}, title = {{A Bifunctional dCTP Deaminase-dUTP Nucleotidohydrolase from the Hyperthermophilic Archaeon Methanocaldococcus jannaschii}}, url = {{http://dx.doi.org/10.1074/jbc.M213010200}}, doi = {{10.1074/jbc.M213010200}}, volume = {{278}}, year = {{2003}}, }