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d-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of d-amino acids. The wild-type DAAO is mainly active on neutral d-amino acids, while basic d-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants...
There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor ζ subunit. This domain...
Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes,...
RNase P is an essential and ubiquitous endonuclease that mediates the maturation of the 5′ ends of all precursor tRNA molecules. The holoenzyme from Dictyostelium discoideum possesses RNA and protein subunits essential for activity, but the exact composition of the ribonucleoprotein complex is still under investigation. Bioinformatic analysis of D. discoideum genome identified seven open reading frames...
Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this,...
Two proteins with phospholipase A 2 (PLA 2 ) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14kDa by SDS-PAGE...
We have previously reported the isolation of a 52,000 M r protein (Pf52) displaying consensus sequences for thiol:disulfide oxidoreductases. Pf52 therefore represents the plasmodial protein disulfide isomerase (PDI). It has been renamed PfPDI and correlates to MAL8P1.17 in the annotated genome of P. falciparum (3D7 strain). Antibodies were raised against recombinant (His) 6 -tagged...
Unlike classical digestive lipases, the scorpion digestive lipase (SDL) has a strong basic character. The SDL activity's optimal pH, when using tributyrin or olive oil as substrate, was 9.0. Added to that, the estimated isoelectric point of the native SDL using the electrofocusing technique, was found to be higher than 9.6. To our knowledge, this is the first report of an animal digestive lipase having...
A depletion in long-chain polyunsaturated ω3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of d-[U- 14 C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and ω3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the ω3-depleted...
Lampetra japonica is representative of the ancient cyclostomota class of animals, and its buccal gland secretion (called lamphredin) is known to act as an anticoagulant. In this study, it was observed by both native-PAGE and SDS-PAGE that the secretion mainly contained two protein bands, buccal gland secretion protein-1 (BGSP-1, 159,909Da) and buccal gland secretion protein-2 (BGSP-2, 25,660Da). The...
The DNA topoisomerases are essential for DNA replication, transcription, recombination, as well as for chromosome compaction and segregation. They may have appeared early during the formation of the modern DNA world. Several families and subfamilies of the two types of DNA topoisomerases (I and II) have been described in the three cellular domains of life (Archaea, Bacteria and Eukarya), as well as...
Candida guilliermondii and human DNA topoisomerases I are inhibited by PL (pyridoxal), PLP (pyridoxal 5′-phosphate) and PLP-AMP (pyridoxal 5′-diphospho-5′-adenosine) (PL<PLP<PLP-AMP).We have recently shown that PLP acted as a competitive inhibitor of C. guilliermondii topoisomerase I, impeding the formation of the cleavable complex from a selective binding to an active site lysine. The targeted...
Mitochondria are the only organelles containing metabolically active DNA besides nuclei. By analogy with the nuclear topoisomerases, mitochondrial topoisomerase activities are probably critical for maintaining the topology of mitochondrial DNA during replication, transcription, and repair. Mitochondrial diseases include a wide range of defects including neurodegeneracies, myopathies, metabolic abnormalities...
Reverse gyrase was discovered more than twenty years ago. Recent biochemical and structural results have greatly enhanced our understanding of their positive supercoiling mechanism. In addition to new biochemical properties, a fine tuning of reverse gyrase regulation in response to DNA damaging agents has been recently described. These data give us a new insight in the cellular role of reverse gyrase...
Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which...
Type IA topoisomerases are enzymes that can modify DNA topology. They form a distinct family of proteins present in all domains of life, from bacteria to archaea and higher eukaryotes. They are composed of two domains: a core domain containing all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain that is highly variable in size and sequence. The latter...
The level of negative DNA supercoiling of the Escherichia coli chromosome is tightly regulated in the cell and influences many DNA metabolic processes including DNA replication, transcription, repair and recombination. Gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a unique ability that arises from the specialized C-terminal DNA wrapping domain of the GyrA...
DNA transaction events occurring during cell life (replication, transcription, recombination, repair, cell division) are always linked to severe changes in the topological state of the double helix. However, since naked DNA almost does not exist in eukaryote nucleus but rather interacts with various proteins, including ubiquitous histones, these topological changes happen in a chromatin context. This...
DNA is not only a nucleotide sequence which allows the binding of regulators but its intrinsic structural properties such as curvature and flexibility are also viewed as playing an active role in the regulation of transcription. Our combination of computer modelling and AFM imaging allow direct access to DNA curvature and flexibility. We have searched for these DNA structural features involved in...
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