Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys 47 and Cys 95 and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4 Å resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). Trypanosomes and Leishmania, the causative agents of several tropical diseases, have a unique thiol redox metabolism that is based on trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine) and the flavoenzyme trypanothione reductase (for reviews, see Refs. 1 and 2). The parasites lack catalases and selenocysteine-containing glutathione peroxidases. 2-Cys-peroxiredoxins and cysteine-containing glutathione peroxidasetype enzymes are responsible for hydroperoxide reduction acting both as tryparedoxin peroxidases (for a recent review, see Ref.3). With NADPH as primary electron source, the reducing equivalents flow via trypanothione and tryparedoxin (Tpx), 2 a distant relative of the thioredoxin protein family, onto the peroxidases which then reduce the hydroperoxide substrates (Scheme 1). In Trypanosoma brucei, the causative agent of African sleeping sickness, both the cytosolic 2-Cys peroxiredoxin and the glutathione peroxidase-type enzymes proved to be essential (4, 5).Classical glutathione peroxidases (GPXs) are selenoproteins. Five distinct enzymes have been characterized in mammals, cytosolic GPX1, gastro-intestinal GPX2, plasma GPX3, phospholipid hydroperoxide peroxidase GPX4, and in humans, GPX6, which is restricted to the olfactory system (6). The high specificity of GPX1 for glutathione as reducing substrate gave the protein family its name (7). In the epididymis of rodents and monkeys, a GPX5 is expressed that contains an active site cysteine instead of the selenocysteine. Recently the structure of human GPX7, also a cysteine homologue, has been solved (PDB code 2P31). Expression of this gene is dramatically down-regulated in breast cancer cells (8). In general, cysteine-containing glutathione peroxidase-type enzymes are widespread in nature, and most of the enzymes, few of which are biochemically characterized, function as thioredoxin peroxidases (for reviews, see Refs. 9 and 10).The selenoenzymes contain a catalytic triad. The selenoate of the peroxidatic selenocysteine is supposed to be stabilized by hydrogen bonds with a Gln and a Trp residue (11, 12). Site-directed mutagenesis (13) and computational studies (14, 15) supported the crucial role of the Gln and Trp residue for catalysis. Both residues ...

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A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., Au and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.

Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.

PAGE 30408:In the left column, the sentence beginning on line 12 from the bottom should read as follows: "In addition, the wild type enzyme obtained here exerted a three times higher specific activity." The sentence beginning on line 5 from the bottom should read as follows: "The apparent second order rate constants (k 1 Ј) of wild type, Glu 97 , and Glu 99 Px III were on the same order of magnitude, whereas the k 2 Ј values of Glu 97 and Glu 99Px III were 10 and 5 times lower than that of the wild type enzyme, respectively (Table 3)." The corrected version of Table 3 is shown below. ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.