FTO, the first RNA demethylase discovered, mediates the demethylation of internal N-methyladenosine (mA) and N, 2-O-dimethyladenosine (mA) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal mA and cap mA in mRNA, internal mA in U6 RNA, internal and cap mA in snRNAs, and N-methyladenosine (mA) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal mA than the ones with cap mA in the tested cells. We also show that FTO can directly repress translation by catalyzing mA tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to mA and mA in mRNA and snRNA as well as mA in tRNA.

Summary Transfer RNA (tRNA) is a central component of protein synthesis and cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and their decreased usage in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new regulatory mechanism of post-transcriptional gene expression.

SummaryTransfer RNA (tRNA) is a central component of protein synthesis and cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N 1 -methyladenosine (m 1 A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and their decreased usage in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new regulatory mechanism of post-transcriptional gene expression. In briefReversible tRNA methylation helps translation respond to nutrient availability.

Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin's regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity-and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB's DNAbinding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.metalloprotein | coregulator | reactive oxygen species (ROS) | oxidative stress | signal transduction activation

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Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual sixelectron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.cofactor biosynthesis | tryptophan radical | heme | posttranslational modification | electron transfer P rotein-based radicals, particularly on tyrosine (Tyr) and tryptophan (Trp) residues, have been implicated in a large number of catalytic and electron transfer reactions in biology (1), including the long-range electron transfer reactions required for photosynthesis (2), respiration (3), and DNA synthesis (4) and repair (5). Aberrant formation of protein radicals during oxidative stress is also of special importance. Evidence for the involvement of protein-based and substrate-based radicals in enzyme-catalyzed reactions has increased substantially in recent years, and expanded our knowledge of the scope of chemical reactions accessible to enzymes. The ability of enzymes to catalyze what were previously thought to be unattainable reactions is exemplified by MauG. The biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH) requires posttranslational modification of two tryptophan residues. MADH from Paracoccus denitrificans is a 119-kDa α 2 β 2 heterotetramer that contains two active sites and two TTQ cofactors derived from residues βTrp57 and βTrp108 (6). MauG is a c-type diheme enzyme that catalyzes the conversion of a MADH precursor (preMADH) that has one oxygen atom already inserted into the βTrp57 indole ring (βTrp57-OH of preTTQ) (7), to mature TTQ-containing MADH (8) (Fig. 1).Catalysis by MauG proceeds via a bis-Fe(IV) redox state, which may be generated by reaction of di-Fe(II) MauG with O 2 or di-Fe(III) MauG with H 2 O 2 (9). Catalytically active crystals of the MauG-preMADH protein complex show that the site of TTQ formation on β-preMADH is 40.1 Å from the MauG highspin heme iron...

[VO(Sal-L-tryp)(H2O)] 1 (where sal-L-tryp = N-salicylidene-L-tryptophanate) was used as a precursor to produce the novel complexes, [VO(Sal-L-tryp)(MeATSC)].1.5C2H5OH 2 (where MeATSC = 9-Anthraldehyde-N(4)-methylthiosemicarbazone), [VO(Sal-L-tryp)(N-Ethhymethohcarbthio)].H2O 3 (where N-Ethhymethohcarbthio = (E)-N-ethyl-2-(4-hydroxy-3-methoxybenzylidene)hydrazinecarbothioamide), and [VO(Sal-L-tryp)(acetylethTSC)].C2H5OH 4 (where acetylethTSC = (E)-N-ethyl-2-(1-(thiazol-2-yl)ethylidene)hydrazinecarbothioamide), by reaction with the respective thiosemicarbazone. The chemical and structural properties of these ligands and complexes were characterised by elemental analysis, ESI MS, FT-IR, UV-visible, ESR, 1H and 13C NMR spectroscopy, and X-ray crystallography. DMSO and DMSO-d6 solutions of compounds 1-4 were oxidised in air to produce vanadium(V) species which were verified by ESI MS and 51V NMR spectroscopy. Anti-cancer properties of compounds 2-4 were examined with three colon cancer cell lines, HTC-116, Caco-2, and HT-29, and also with non-cancerous colonic myofibroblasts, CCD18-Co. Compounds 2-3 exhibited less inhibitory effects in the CCD-18Co cells, indicating a possible cytotoxic selectivity towards colon cancer cells. In general, those compounds which exhibited anti-proliferative activity on cancer cells, but did not affect non-cancerous cells, may have a potential in chemotherapy.

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.