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D C Rees

Publications and source records attributed to D C Rees.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structure of a thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus.

The 2.3 A resolution crystal structure of a [2Fe-2S] cluster containing ferredoxin from Aquifex aeolicus reveals a thioredoxin-like fold that is novel among iron-sulfur proteins. The [2Fe-2S] cluster is located near the surface of the protein, at a site corresponding to that of the active-site disulfide bridge in thioredoxin. The four cysteine ligands are located near the ends of two surface loops. Two of these ligands can be substituted by non-native cysteine residues introduced throughout a stretch of the polypeptide chain that forms a protruding loop extending away from the cluster. The presence of homologs of this ferredoxin as components of more complex anaerobic and aerobic electron transfer systems indicates that this is a versatile fold for biological redox processes.

Amino Acid Sequence↗
PubMed2000

Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states.

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

Crystallography, X-Ray↗
PubMed2000

Conformational flexibility of B-DNA at 0.74 A resolution: d(CCAGTACTGG)(2).

The affinity and specificity of a ligand for its DNA site is a function of the conformational changes between the isolated and complexed states. Although the structures of a hydroxypyrrole-imidazole-pyrrole polyamide dimer with 5'-CCAGTACTGG-3' and the trp repressor recognizing the sequence 5'-GTACT-3' are known, the baseline conformation of the DNA site would contribute to our understanding of DNA recognition by these ligands. The 0.74 A resolution structure of a B-DNA double helix, 5'-CCAGTACTGG-3', has been determined by X-ray crystallography. Six of the nine phosphates, two of four bound calcium ions and networks of water molecules hydrating the oligonucleotide have alternate conformations. By contrast, nine of the ten bases have a single, unique conformation with hydrogen atoms visible in most cases. The polyamide molecules alter the geometry of the phosphodiester backbone, and the water molecules mediating contacts in the trp repressor/operator complex are conserved in the unliganded DNA. Furthermore, the multiple conformational states, ions and hydration revealed by this ultrahigh resolution structure of a B-form oligonucleotide are potentially general considerations for understanding DNA-binding affinity and specificity by ligands.

Bacterial Proteins↗
PubMed2000

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove.

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.

Adenine↗
PubMed2000

Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli.

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.

Bacterial Proteins↗
PubMed2000

Analyzing your complexes: structure of the quinol-fumarate reductase respiratory complex.

The integral membrane protein complex quinol-fumarate reductase catalyzes the terminal step of a major anaerobic respiratory pathway. The homologous enzyme succinate-quinone oxidoreductase participates in aerobic respiration both as complex II and as a member of the Krebs cycle. Last year, two structures of quinol-fumarate reductases were reported. These structures revealed the cofactor organization linking the fumarate and quinol sites, and showed a cofactor arrangement across the membrane that is suggestive of a possible energy coupling function.

Animals↗
PubMed2000

Nitrogenase: standing at the crossroads.

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, which is central to the process of biological nitrogen fixation. Recent progress towards establishing the mechanism of action of this complex metalloenzyme reflects the contributions of a combination of structural, biochemical, spectroscopic, synthetic and theoretical approaches to a challenging problem with implications for a range of biochemical and chemical systems.

Adenosine Triphosphate↗
PubMed2000

Structure of a photoactive rhodium complex intercalated into DNA.

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.

Base Sequence↗
PubMed2000

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group.

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.

Acute Disease↗
PubMed2000

Are aberrant BCR--ABL transcripts more common than previously thought?

We report the use of multiplex polymerase chain reaction (PCR), using 4% polyacrylamide gel electrophoresis (PAGE) for the detection of BCR-ABL transcripts in Philadelphia-positive disease. Three out of 50 cases [two out of 37 chronic myeloid leukaemia (CML), one out of 13 acute lymphoblastic leukaemia (ALL)] possessed rare breakpoints; an e19a2 and e13a3 in CML and an e1a3 in the ALL. We suggest that multiplex PCR using 4% PAGE and optimized for smaller transcript detection may lead to a higher detection rate of rare BCR-ABL breakpoints. Multiplex PCR, however, failed to distinguish e13a2 from e1a3 transcripts. Finally, the presence of e13a3 in CML supports the view that abl exon 2 sequences are unnecessary for the pathogenesis of 'classic' CML.

Chromosome Breakage↗
PubMed1999

Why are hemoglobin F levels increased in HbE/beta thalassemia?

To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of beta thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/beta thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the alpha/gamma, beta(E)/gamma, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/beta thalassemia, and other beta thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

Adolescent↗
PubMed1999

Structural bioenergetics and energy transduction mechanisms.

Life depends on transduction processes that couple cellular metabolism to environmental energy sources such as light or reduced compounds. These primary energy sources must be efficiently converted into forms that can be utilized by cells for biosynthesis, motility, transport, regulation, and other metabolic functions. In recent years, there has been an explosive increase in the determination of structures for proteins mediating energy transduction processes. These developments provide the opportunity to evaluate the structural basis for the efficient coupling of two energetic processes, which defines the area of structural bioenergetics. Here, we present some general features of energy transduction processes, including arguments that effective coupling of two processes by a transduction protein occurs by way of conformational states that are common to the catalysis of each process. This is illustrated by examples from the nucleotide switch family of proteins, with emphasis on the nitrogenase system where ATP hydrolysis is coupled to an electron transfer reaction.

Animals↗
PubMed1999

Structure of the Escherichia coli fumarate reductase respiratory complex.

The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc1.

Aerobiosis↗
PubMed1999

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus: the 1.85 A resolution crystal structure and its mechanistic implications.

Crystal structures of formaldehyde ferredoxin oxidoreductase (FOR), a tungstopterin-containing protein from the hyperthermophilic archaeon Pyrococcus furiosus, have been determined in the native state and as a complex with the inhibitor glutarate at 1.85 A and 2. 4 A resolution, respectively. The native structure was solved by molecular replacement using the structure of the homologous P. furiosus aldehyde ferredoxin oxidoreductase (AOR) as the initial model. Residues are identified in FOR that may be involved in either the catalytic mechanism or in determining substrate specificity. The binding site on FOR for the physiological electron acceptor, P. furiosus ferredoxin (Fd), has been established from an FOR-Fd cocrystal structure. Based on the arrangement of redox centers in this structure, an electron transfer pathway is proposed that begins at the tungsten center, leads to the (4Fe:4S) cluster of FOR via one of the two pterins that coordinate the tungsten, and ends at the (4Fe:4S) cluster of ferredoxin. This pathway includes two residues that coordinate the (4Fe:4S) clusters, Cys287 of FOR and Asp14 of ferredoxin. Similarities in the active site structures between FOR and the unrelated molybdoenzyme aldehyde oxidoreductase from Desulfovibrio gigas suggest that both enzymes utilize a common mechanism for aldehyde oxidation.

Aldehyde Oxidoreductases↗
PubMed1999

Minisatellite mutational processes reduce F(st) estimates.

We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2,689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (F(st)) between populations is less than 8%, which is lower than comparative estimates of between 10%-15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing F(st) estimates from minisatellite data because, first, the lowest F(st) estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, F(st) declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple step-wise mutation models.

Alleles↗