Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54°C, 23°C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg 2؉ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

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confidence: 76%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

“…This spacer crosses over to the substrate bound at the neighboring subunit to form the closed, catalytically competent enzyme conformer. The feline immunodeficiency virus dUTPase structure (56) reveals that spacer shortening may seriously compromise formation of the closed conformer (Fig. 7B).…”

Section: Binding Of Zn 2ϩ To the Zn-knuckle Motifs Within Nc Wasmentioning

confidence: 99%

dUTPase and Nucleocapsid Polypeptides of the Mason-Pfizer Monkey Virus Form a Fusion Protein in the Virion with Homotrimeric Organization and Low Catalytic Efficiency

Barábas

Rumlová

Erdei

et al. 2003

“…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.…”

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confidence: 99%

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

Johansson

Fano

Bynck

et al. 2005

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg 115 and Glu 138 , were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wildtype E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg 2؉ , and the mutant enzyme also with the substrate dCTP and Mg 2؉ . The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg 2؉ that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.The main source of dUMP, the precursor for dTTP in the Gram-negative bacteria Escherichia coli and Salmonella typhimurium, is obtained via a pathway where dCTP is deaminated by dCTP deaminase (EC 3.5.4.13) to yield ammonia and dUTP that subsequently is hydrolyzed by dUTPase to generate dUMP and pyrophosphate (1). In contrast, Gram-positive bacteria and eukaryotic organisms synthesize dTTP from dUMP obtained by deamination of dCMP by the zinc-containing enzyme dCMP deaminase (2). 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.One very interesting feature of the dCTP deaminase is that the deamination reaction proceeds without aid from a metal cofactor. Other nucleobase or nucleoside deaminases such as cytosine deaminase (6, 7), cytidine deaminase (8), adenosine deaminase, (9) and adenine deaminase (10) all require a catalytic m...