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

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-Å 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 ␤-barrel surrounded by two ␤-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-residuelong 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 Ser 118 , Arg 122 , Thr 130 , and Glu 145 . These water molecules are potential nucleophile candidates in the deamination reaction.dUMP is the origin of thymidine precursors for DNA in all organisms. It is the substrate of thymidylate synthase that catalyzes the formation of dTMP (1). The main pathway for dUMP generation differs markedly between different organisms. dUMP is produced on the monophosphate level by the action of dCMP deaminase in eukaryotes and Gram-positive bacteria, whereas in Gram-negative bacteria such as Escherichia coli and some Archaea, dCTP is deaminated and dephosphorylated. dCTP deaminase (EC 3.5.4.13) and dUTPase (EC 3.6.1.23) catalyze the two consecutive steps where dUMP is formed in E. coli (dCTP 3 dUTP 3 dUMP). The hyperthermophilic archaeon Methanocaldococcus jannaschii produces a bifunctional dCTP deaminase-dUTPase (DCD-DUT) 1 with the ability to catalyze both reactions ( Fig. 1) (2, 3). The gene that encodes this enzyme was first annotated as coding for a dCTP deaminase, but enzymatic studies revealed the bifunctionality, which implies that dUTP is never released from the enzyme.dUTP is also formed upon spontaneous deamination of dCTP, and the reaction catalyzed by dUTPase is therefore essential in keeping the cellular concentration of dUTP low to suppress misincorporation of uracil into DNA. If such an event would happen, DNA is restored by an inherent repair mechanism. However, when dUTPase is deficient, the organism will not survive (4, 5). The dUTPases exist in different oligomeric forms. The homotrimeric form of dUTPases is by far the most common, but there are also mo...

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…1 An additional enzymatic activity, deamination of dCTP (deoxycytidine triphosphate) to dUTP, may also be carried out by bifunctional dUTPases in Archaea [2][3][4][5] and also in Mycobacterium tuberculosis (M. tuberculosis), which also has a monofunctional dUTPase. 6 The dUTPases comprise two structurally different classes: 7 first, the β sheet−type dUTPases, possessed by most organisms, employ a one-metal ion catalytic mechanism, [8][9] and usually form a homotrimer 10 or a monomer, 11 while the second class, the α helical-type dUTPases, possessed by kinetoplastids, 12 form dimers and employ a two-metal ion catalytic mechanism.…”

Structures of dCTP Deaminase from Escherichia coli with Bound Substrate and Product

“…Recently, a bifunctional enzyme from the archaeon Methanocaldococcus jannaschii has been identified (3,4) that possesses both the dCTP deaminase and dUTPase activities in one polypeptide suggesting that at least in some Archaea, dCTP serves as a source for dUMP. The structure of this archaeal enzyme is now known and the subunit shares an overall fold with dUTPases as well as the organization of subunits in a trimer (5). In the present work we demonstrate that dCTP deaminase from E. coli is yet another member of this family of enzymes, even though significant differences are found in the C-terminal stretch that closes the active site upon catalysis.…”

“…Structural Similarity-The structure of E. coli dCTP deaminase show the greatest similarity to the previously determined structure of the bifunctional dCTP deaminase-dUTPase from M. jannaschii (5,36), as demonstrated by the possibility to use parts of this structure as a search model and obtaining a correct molecular replacement solution. One subunit of E. coli dCTP deaminase (chain A, E138A⅐dCTP⅐Mg 2ϩ ) and the bifunctional dCTP deaminase-dUTPase from M. jannaschii (PDB code 1OGH; chain A) superimpose with a root mean square deviation of 1.30 Å for 140 C␣ atoms as determined using default parameters in the program O (26) (Fig.…”

“…Like the bifunctional enzyme, dCTP deaminase is also similar to homotrimeric dUTPases (Fig. 5, A and B) (5). 98 C␣ atoms from E. coli dUTPase (PDB code 1DUD) superimpose, as above, with chain A from E. coli dCTP deaminase (E138A⅐dCTP⅐Mg 2ϩ ) with a root mean square deviation of 1.80 Å (21% identity).…”

“…Trp 131 is stacked with the deoxyribose ring in an equivalent way to the tyrosine or phenyl alanine residues found in dUTPase structures (32)(33)(34)(35) and the bifunctional dCTP deaminasedUTPase from M. jannaschii (5,36). The enzyme's discrimination against CTP appears to be a result of the large, hydrophobic side chain of Trp 131 that leaves no room for a hydroxyl group at position 2 on the ribose ring.…”