Rebecca Persson

Homotrimeric dUTPases; structural solutions for specific recognition and hydrolysis of dUTP.

Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis. The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a beta-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues. All dUTPases studied require the presence of a divalent metal ion, preferably Mg(2+), for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase. Structure-function studies are essential if the properties of dUTPases are to be understood fully in relation to their biological role. In this review the structural arrangement of the homotrimeric dUTPases is discussed in the context of active site geometry, achievement of specificity and subunit interactions.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non‐hydrolyzable 2′‐deoxyuridine 5′‐(α,β‐imido)triphosphate (α,β‐imido‐dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+‐dependent, does not appear with dUDP instead of α,β‐imido‐dUTP and is not elicited if the flexible C‐terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C‐terminal arm in α,β‐imido‐dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg‐141. Near UV CD of ligand‐enzyme complexes reveals a characteristic difference in the microenvironments of enzyme‐bound dUDP and α,β‐imido‐dUTP, a difference not observable in C‐terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C‐terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

Atomic resolution structure of Escherichia coli dUTPase determined ab initio

Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05 A resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45 A from a native crystal were also collected and the 100 K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor-substrate-analogue complexes of this protein at very high resolution.

dUTPase from Escherichia coli; high-level expression and one-step purification

ABSTRACT The dut gene, which encodes Escherichia coli deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500 mg per litre of bacterial culture, a significant increase compared to previously employed methods.

The structure of Bacillus subtilis SPβ prophage dUTPase and its complexes with two nucleotides

dUTPases are housekeeping enzymes which catalyse the hydrolysis of dUTP to dUMP in an ion-dependent manner. Bacillus subtilis has both a genomic and an SPβ prophage homotrimeric dUTPase. Here, structure determination of the prophage apoenzyme and of its complexes with dUDP and dUpNHpp-Mg(2+) is described at 1.75, 1.9 and 2.55 Å resolution, respectively. The C-terminal extension, which carries the conserved motif V, is disordered in all three structures. Unlike all other trimeric dUTPases for which structures are available, with the exception of the Bacillus genomic enzyme, the aromatic residue covering the uridine and acting as the Phe-lid is close to motif III in the sequence rather than in motif V. This is in spite of the presence of an aromatic amino acid at the usual Phe-lid position in motif V. The alternative position of the Phe-lid requires a reconsideration of its role in the catalytic cycle of the enzyme. In the dUpNHpp-Mg(2+) complex a water can be seen at the position expected for nucleophilic attack on the α-phosphate, in spite of motif V being disordered. Differences in the active site between the free enzyme and the dUDP and dUpNHpp-Mg(2+) complexes shows that the triphosphate moiety needs to be in the gauche conformation to trigger the conformational changes that can be seen in both B. subtilis dUTPases.

Challenges of patient-focused care: Nurses’ descriptions and observations before and after intervention

The concept of patient-focused care aims to provide an environment in which the healthcare team focuses on the individual patient’s needs. In order to increase our understanding of how nurses perceive and conduct patient-focused care, the issue needs to be studied in various contexts. The aims of the study were to explore nurses’ descriptions of their patient-focused care, what took place during observed situations including the time spent, before and after the change of design from a more traditional to a single-bed hospital in Sweden. Non-participant observations with follow-up interviews were carried out. Data were analysed using qualitative content analysis. Three categories emerged from the analysis: Barriers to being close to the patient, Desire to be close to the patient and The influence of environment on caring. The theme Presence or absence was interpreted as the latent meaning. The conclusion was that being present is crucial in nursing when providing compassionate and effective nursing care.

Possibilities for Patients with Elevated Blood Pressure to Achieve Blood Pressure Control without Affecting Quality of Life (the PEQ study): A Study Protocol

Several interventions on adherence have been tested in hypertension care but still the number of patients with well controlled blood pressure is not increasing. The aim is to get a deeper understanding of the patients’ reasons for not following their treatment as a base for in collaboration with the patients, developing effective interventions. A mixed methods design is to be used. Patients with hypertension who have considered changing lifestyle will be interviewed individually about their reasons for changing or not changing lifestyle and for taking or not taking medicines. Other patients, both those who do and those who do not have well-controlled blood pressure, treated at health centres and hospital clinics, will be asked to fill in instruments. The Exercise of Self Care Agency instrument gives information about the patients’ ability to perform self-care (change lifestyle) and the SF-36 is about health-related quality of life. Finally, patients will be asked to participate in focus-group interviews about how they want to be treated and what would be of help for them to achieve blood pressure control. From the findings we will create intervention/interventions without negative impact on quality of life together with the patients. These interventions are to be carried out and evaluated in real practice with patients with hypertension and other significant persons or health care personnel that may be involved.