The C‐terminus of dUTPase: observation on flexibility using NMR

Nord, Johan; Nyman, Per-Olof; Larsson, Gunilla; Drakenberg, Torbjörn

doi:10.1016/s0014-5793(01)02257-8

Cited by 20 publications

(24 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This conformational change is realized by intricate subunit interactions as the C terminus of one subunit crosses over its neighboring subunit and adopts an ordered conformation to contact the substrate bound in the active site of the third subunit. In the bacterial enzyme, detailed functional investigations (2,30,33,34), in agreement with the crystal structure in complex with the nucleotide dUDP (28), indicate that the ␥ phosphate of the substrate is indispensable in inducing the closed conformer, and after catalytic cleavage the active site pops open. In the case of the human enzyme, the crystal structure suggests the retaining of the closed conformer even with the product dUMP (24), but there are no functional data published.…”

supporting

confidence: 55%

“…Conformational Shifts in the C Terminus Are Induced Differently in Drosophila and E. coli dUTPases-Detailed studies from several laboratories unanimously indicated that the ordering of the C terminus of E. coli dUTPase requires interaction with the complete triphosphate chain of the substrate (28,30,34). In the present work, experimental evidence from limited proteolysis, CD spectroscopy, and DSC argue in agreement that dUMP and dUDP are also capable of inducing a significant conformational change (an increment in ordered protein conformation) upon binding to D. melanogaster dUTPase.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

supporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

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

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The sequence of the C-terminal motif V shares a limited number of features with those of the P-loop motifs (Fig. 1A) (3, 4) present in a large number of ATPase and GTPase enzyme families known as P-loop NTPases including kinases, cytoskeleton and DNA motors, membrane pumps, and transporters.It has been shown that the C terminus of dUTPase is necessary for dUTP hydrolysis but not for nucleotide binding or structural integrity (3,(5)(6)(7). Surprisingly, the active site architecture with bound substrate in the crystal structure of the C-terminally truncated inactive enzyme is identical to that of the wild-type (WT) (7).…”

mentioning

confidence: 99%

“…It has been shown that the C terminus of dUTPase is necessary for dUTP hydrolysis but not for nucleotide binding or structural integrity (3,(5)(6)(7). Surprisingly, the active site architecture with bound substrate in the crystal structure of the C-terminally truncated inactive enzyme is identical to that of the wild-type (WT) (7).…”

mentioning

confidence: 99%

Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination

Pecsi

Szabó

Adams

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTPase mutants combined with previous structural studies indicate that (i) H-bond interactions between the γ-phosphate and the P-loop-like motif V promote the catalytically competent conformation of the reaction center at the α-phosphate group; (ii) these interactions accelerate the chemical step of the kinetic cycle and that (iii) hydrolysis occurs very slowly or not at all in the absence of the γ-phosphate-motif V interactions, i.e., in dUDP, dUDP.BeFx, or in the motif V-deleted mutant. The physiological role of dUTPase is to set cellular dUTP∶dTTP ratios and prevent injurious uracil incorporation into DNA. Based upon comparison with related pyrophosphate generating (d)NTPases, we propose that the unusual use of a P-loop-like motif enables dUTPases to achieve efficient catalysis of dUTP hydrolysis and efficient discrimination against dUDP at the same time. These specifics might have been advantageous on the appearance of uracil-DNA repair. The similarities and differences between dUTPase motif V and the P-loop (or Walker A sequence) commonly featured by ATP-and GTPases offer insight into functional adaptation to various nucleotide hydrolysis tasks.NTP hydrolysis | nucleotide discrimination | dUTP pyrophosphatase | Walker A motif | evolutionary adaptation I t is an intriguing question how pyrophosphate generating nucleotide hydrolases distinguish between (d)NDP and (d)NTP both containing the α-β phosphoanhydride bond to be hydrolyzed. In the present paper, we investigate two fundamental questions related to the nucleotide pyrophosphatase enzymatic activity exhibited by the enzyme dUTPase and by other pyrophosphatases: (i) the mechanism of discrimination between nucleoside di-and triphosphate ligands; (ii) the potential contribution of a P-loop-like motif to such discrimination. The enzyme dUTPase naturally evoked these questions as it specifically performs the hydrolysis of dUTP between the α-β phosphates with no further coupled reactions and it contains a P-loop-like motif (Fig. 1A). In addition, the structural comparison between Mg:dUDP-and Mg:dUTP analog-bound enzymes does not offer a straightforward explanation to why dUDP is not hydrolyzed because the scissile bond and the nucleophile adopt the same conformation in both (Fig. 1B and in stereo in Fig. S1A, the sole Mg:dUDPcomplexed structure is superposed with a human Mg:dUPNPP complex).dUTPase hydrolyzes dUTP to yield dUMP (a precursor for dTTP biosynthesis) and pyrophosphate (PP i ). The action of dUTPase is the only known direct mechanism to minimize uracil incorporation into DNA (1). Most dUTPases are homotrimers and confer three active sites. The substrate in each active site is bound by conserved sequence motif...

show abstract

“…Correspondingly, amino acid sequences of the ordered and disordered segments of the C-terminal portions of the E. coli and human enzymes are compared in Table 1. In the E. coli enzyme the C-terminal region is ordered only on binding 2Ј-deoxyuridine with a 5Ј-triphosphate group in the presence of a divalent metal ion (9,10,14). Because ordering of the C-terminal region of the polypeptide chain has been shown to be required for catalytic action by the enzyme (9-11), an approach must be adopted for structural analysis that can be applied to macromolecules in solution.…”

mentioning

confidence: 99%

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Mustafi

Békési

Vértessy

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of k cat and kcat͞Km were 4.5 ؎ 0.1 s ؊1 and 0.49 ؎ 0.1 ؋ 10 6 M ؊1 ⅐s ؊1 in the absence of divalent metal ions, 14.7 ؎ 2.2 s ؊1 and 25.1 ؎ 7.4 ؋ 10 6 M ؊1 ⅐s ؊1 in the presence of Mg 2؉ or Mn 2؉ , and 24.2 ؎ 3.6 s ؊1 and 2.4 ؎ 0.7 ؋ 10 6 M ؊1 ⅐s ؊1 when supported by VO 2؉ or bis(acetylacetonato)oxovanadium(IV). Binding of VO 2؉ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg 2؉ . Electron paramagnetic resonance spectra of the ternary enzyme-VO 2؉ -chelatedUDP complex revealed a pattern of 31 P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO 2؉ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO 2؉ . We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO 2؉ and the VO 2؉ -chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the Cterminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.C ontrary to ordinary expectation, the nucleic acid uracil can be incorporated into DNA in the cell because DNA polymerases do not distinguish between thymine and uracil. The most important cellular vehicle for hindering this process is the enzyme dUTP pyrophosphatase (dUTPase) (1, 2). This enzyme hydrolyzes dUTP into dUMP and pyrophosphate, thereby removing dUTP from the DNA biosynthetic pathway. Although several high to medium resolution x-ray studies of dUTPases from human (3), bacterial (4-6), and viral (7, 8) sources have been reported, there is no understanding of the structural basis of the catalytic mechanism. The C-terminal portion of each subunit of this trimeric enzyme is partly disordered in crystals and does not contribute to the electron density map. Furthermore, the binding site of Mg 2ϩ in the active site has not been identified. In the crystal structure of the equine infectious anemia virus (EIAV) enzyme, a Sr 2ϩ ion bound to both the ␣-and ␤-phosphate groups of dUDP has been localized (7), but in a position that would sterically prevent nucleophilic attack by a hydrolytic water molecule. Nonetheless, the catalytic importance of the C-terminal residues and the dependence of dUTPase action on Mg 2ϩ have been amply demonstrated by studies of the enzyme in solution (9-12). Because dUTPase has acquired increasing attention as a potential target enzyme in cancer and antiretroviral chemot...

show abstract

The C‐terminus of dUTPase: observation on flexibility using NMR

Cited by 20 publications

References 27 publications

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

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

Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Contact Info

Product

Resources

About