Modified nucleotides are present in many RNA species in all Domains of Life. While the biosynthetic pathways of such nucleotides are well studied, much less is known about the degradation of RNAs and the return to the metabolism of modified nucleotides, their respective nucleosides or heterocyclic bases. Using an E. coli uracil auxotroph, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. We show that a gene encoding a protein consisting of previously uncharacterized Domain of Unknown Function 523 (DUF523) is responsible for such phenotype. We have purified this recombinant protein and demonstrated that it contains a FeS cluster. The substitution of cysteines, which have been predicted to form such clusters, with alanines abolished the growth phenotype. We conclude that DUF523 is involved in the conversion of 2-thiouracil into uracil in vivo.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity called TudS. We report here the crystal structure of TudS, refined at 1.5 Å resolution, which harbors a [4Fe-4S] cluster, bound by three cysteines only. Incubation of TudS crystals with 4thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information together with modeling studies allow us to propose a mechanism for the unprecedented nonredox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and ␣-ketoglutarate. Pyridine and its derivatives are ubiquitous in nature. The pyridine ring is found in alkaloids (e.g., nicotine, actinidine), coenzymes [NAD(P)H, pyridoxal], and man-made solvents, pesticides, and herbicides (e.g., paraquat). Hydroxypyridines are common intermediate metabolites produced during microbial biodegradation of various N-heterocycles (pyridine, nicotine, picoline, 2,6-dipicolinic acid) (1-3).It has previously been reported that Arthrobacter crystallopoietes, Arthrobacter pyridinolis, and Arthrobacter viridescens (4), Achromobacter sp. strain G2 (5), and Nocardia sp. strain PNO (6) use 2-hydroxypyridine (2HP) as a sole carbon and energy source. Through more than 50 years of investigation of pyridine ring metabolism, many intermediates have been identified and metabolic pathways have been proposed. However, the genes and enzymes responsible for 2HP biodegradation have seldom been reported.In Achromobacter sp. G2, 2HP is metabolized via the maleamate pathway (5) (Fig. 1). No enzymes responsible for the initial hydroxylation step of 2HP leading to the formation of 2,5-dihydroxypyridine (2,5DHP) have been reported to date. Nevertheless, the degradation of 2,5DHP, an intermediate of nicotinic acid metabolism, has been fully investigated by Jiménez et al. (7), and all genes encoding the enzymes involved in the maleamate pathway have been identified and characterized (7).Arthrobacter crystallopoietes, A. pyridinolis, and A. viridescens (4) and Arthrobacter sp. strain PY22 (8) produce a blue pigment (nicotine blue) in the medium when grown on 2HP. The nicotine blue has been shown to be a 4,5,4=,5=-tetrahydroxy-3,3=-diazadiphenoquinone-(2,2=) (9) that is an autoxidation product of 2,3,6-trihydroxypyridine (THP). THP can be synthesized via hydroxylation of 2,5DHP, 2,3-dihydroxypyridine (2,3DHP), or 2,6-dihydroxypyridine (2,6DHP); however, only the 2,6DHP 3-hydroxylase, which is involved in the biodegradation of nicotine by Arthrobacter nicotinovorans, has been identified to date (10).We have previously reported that HpyB monooxygenase from Arthrobacter sp. PY22 is sufficient for the conversion of 2HP to THP (8). Since no reaction intermediates have been detected, a consecutive two-st...

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug.