Structure of the Bifunctional dCTP Deaminase-dUTPase from Methanocaldococcus jannaschii and Its Relation to Other Homotrimeric dUTPases

Eva Johansson,Olof Björnberg,Per Olof Nyman,Sine Larsen

doi:10.1074/jbc.m304361200

Eva Johansson, Olof Björnberg + Show 2 more

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https://doi.org/10.1074/jbc.m304361200

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Abstract

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.

Highlights

The bifunctional dCTP deaminase-dUTPase (DCDDUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA
DUTP is formed upon spontaneous deamination of dCTP, and the reaction catalyzed by dUTPase is essential in keeping the cellular concentration of dUTP low to suppress misincorporation of uracil into DNA
In the Ramachandran plot there are no nonglycine residues in disfavored regions except for residues Lys20 and Pro21, which form a cis-peptide bond. This proline residue is strongly conserved among dCTP deaminase amino acid sequences (Fig. 5, supplementary material)

Summary

EXPERIMENTAL PROCEDURES

Crystallization—The MJ0430 gene from M. jannaschii was expressed in E. coli and purified as described in Bjornberg et al [2]. The heavy atom derivative crystal was obtained by the addition of 2 mM lead acetate in the mother liquor. The wavelength of the data collection (1.098 Å) was not ideal for obtaining an optimal anomalous signal from lead, but, the positions of two lead atoms were found with the program SOLVE [19], which may be attributed to the high redundancy and accuracy of the data. The phases were extended from 2.5-Å resolution (lead derivative) to 1.88-Å resolution (native data) using the ARP-wARP program [21], and a free atom model was produced. From this model, 97% of the 356 amino acid residues contained in the final model could be automatically traced by ARP-wARP. Ϳdennis/). The sequence alignment in the supplementary material (Fig. 5) was prepared with T-Coffee [27]

RESULTS

TABLE II Refinement statistics

DISCUSSION