A Bifunctional dCTP Deaminase-dUTP Nucleotidohydrolase from the Hyperthermophilic Archaeon Methanocaldococcus jannaschii

Olof Björnberg,Jan Neuhard,Per Olof Nyman

doi:10.1074/jbc.m213010200

Olof Björnberg, Jan Neuhard + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m213010200

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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 alpha- and beta-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 micro m). 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 micro m) 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.

Highlights

The biosynthesis of the thymidine nucleotide precursor for DNA occurs in all organisms by the reductive transfer of a methylene group from N5,N10-methylene tetrahydrofolate to dUMP, catalyzed by thymidylate synthases [1, 2]
Analysis of the nucleotide sequences of the genes indicated that they harbor a large number of codons rarely used in E. coli
We have shown that the MJ0430 protein of M. jannaschii is a bifunctional enzyme catalyzing two chemical reactions: (i) deamination of the cytosine ring of dCTP and (ii) hydrolysis of the phosphoanhydride bond between the ␣- and ␤-phosphates, yielding dUMP as the end product

Summary

EXPERIMENTAL PROCEDURES

Materials [3H]dUTP and [3H]dCTP were from Amersham Biosciences. Unlabeled nucleotides Q-Sepharose and Red-Sepharose were from Pharmacia Corp. The two genes MJ1102 and MJ0430 were amplified by PCR from genomic M. jannaschi DNA obtained from the American Type Culture Collection (ATCC 43067) with primers introducing restriction endonuclease sites for NdeI and BamHI before the start codon and after the stop codon, respectively. After purification on QIAquick mini columns (Qiagen) and digestion with NdeI and BamHI, the fragments (514 and 638 bp) were cloned into plasmid pET-3a [15], yielding pET-3a/MJ1102 and pET-3a/ MJ0430. DNA sequencing of both strands of the cloned inserts revealed no differences when compared with the published sequences [16]. The two constructs were transformed into E. coli BL21(DE3) and BL21(DE3)/pRI952. Plasmid pRI952 [17] carries genes for the rare tRNAIle (AUA) and tRNAArg (AGA and AGG) codons and for resistance to chloramphenicol (CmR)

Small Scale Expression

Purification of Protein

Enzyme Assays

RESULTS

Construct dUTPase activitya

DISCUSSION