We designed and created nanofabricated quartz cylinders well suited for torque application and detection in an angular optical trap. We made the cylinder axis perpendicular to the extraordinary axis of the quartz crystal and chemically functionalized only one end of each cylinder for attachment to a DNA molecule. We directly measured the torque on a single DNA molecule as it underwent a phase transition from B-form to supercoiled P-form.

In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs, we identified a total of four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3(2) that shared low overall identity to CDO (13 to 21%) but nevertheless conserved important active-site residues. These four proteins were heterologously expressed and purified to homogeneity by a single-step immobilized metal affinity chromatography procedure. The ability of these proteins to oxidize cysteine to cysteine sulfinic acid was then compared against recombinant rat CDO. The kinetic data strongly indicate that these proteins are indeed bona fide CDOs. Phylogenetic analyses of putative bacterial CDO homologs also indicate that CDO is distributed among species within the phyla of Actinobacteria, Firmicutes, and Proteobacteria. Collectively, these data suggest that a large subset of eubacteria is capable of cysteine sulfoxidation. Suggestions are made for how this novel pathway of cysteine metabolism may play a role in the life cycle of the eubacteria that have it.Cysteine is an indispensable amino acid for all forms of life. It serves as a precursor for protein synthesis, and the unique chemistry of its free thiol group forms the active moiety of several essential metabolites such as coenzyme A, glutathione and its derivatives, and mycothiol. Because many basic cellular processes are dependent upon a steady supply of cysteine, there has been extensive work to identify in both prokaryotes and eukaryotes the pathways that ultimately determine the steady-state free intracellular cysteine pool (26). The generic pathways that increase the levels of free intracellular cysteine are those of de novo biosynthesis and transport of preformed cysteine from the extracellular milieu. On the other hand, the pathways responsible for depleting free cysteine include those that incorporate cysteine into other molecules and those that physically degrade the cysteine molecule.Among the specific pathways that contribute to the metabolic economy of intracellular cysteine, one of the major differences between eukaryotes and prokaryotes has been believed to be the capacity to degrade cysteine to cysteine sulfinic acid (21). This reaction, catalyzed by the Fe 2ϩ -dependent enzyme cysteine dioxygenase (CDO; EC 1.13.11.20), irreversibly oxidizes the sulfhydryl group of cysteine (Fig. 1). CDO shows a high degree of specificity for cysteine since structurally related thiols are neither substrates for oxidation nor competitive inhibitors of activity (31). In mammals-eukaryotes for wh...

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNAmediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.There are many different processes in mammalian cells that result in the oxidation of thiol groups. Because of their reactivity, free sulfhydryl groups are highly susceptible to oxidation that results in the formation of disulfides, sulfenates, sulfinates, and sulfonates. Many of these reactions occur nonenzymatically, principally as a consequence of adventitious free radicals arising from aerobic respiration. Nevertheless, there are a small number of thiol oxidation reactions that are known to occur directly via enzymatic catalysis. The enzymes that catalyze these reactions show a high degree of substrate specificity and confer to cells the advantage of being able to precisely regulate the level of a particular reduced thiol.One interesting subset of the enzymes capable of specifically oxidizing free sulfhydryl groups are the thiol dioxygenases. In mammals this family comprises only two known proteins: cysteine dioxygenase (CDO, 3 EC 1.13.11.20) and cysteamine dioxygenase (EC 1.13.11.19). CDO adds two atoms of oxygen to free cysteine to yield cysteine sulfinic acid, whereas cysteamine dioxygenase adds two atoms of oxygen to free cysteamine (2-aminoethanethiol) to form hypotaurine (Fig. 1). The activities for these two proteins were first reported in mammalian tissues almost 40 years ago (1, 2). Since that time, howev...

The common reactions of dioxygen, superoxide and hydroperoxides with thiolates are thought to proceed via persulfenate intermediates, yet these have never been visualized. Here we report a 1.4 Å resolution crystal structure of the Fe2+-dependent enzyme cysteine dioxygenase (CDO) containing this putative intermediate trapped in its active site pocket. The complex raises the possibility that, distinct from known dioxygenases and proposed CDO mechanisms, the Fe-proximal oxygen atom may be involved in the primary oxidation event to yield a unique three-membered Fe-S-O cyclic intermediate. A non-polar environment of the distal oxygen would facilitate isomerization of the persulfenate to the sulfinate product.