Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs.

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

Structure of Urocanate Hydratase from the protozoan Trypanosoma cruzi.

The enzyme Urocanate Hydratase (UH) participates in the catabolic pathway of L-histidine. Trypanosoma cruzi Urocanate Hydratase (TcUH) is identified as a therapeutic molecular target in the WHO/TDR Targets Database. We report the 3D structure determination and number of features of TcUH, and compared it to other few available bacterial UH structures. Each monomer presents two domains and one NAD+ molecule. Superpositions revealed differences in the relative orientation of domains within monomers, such that TcUH monomer A resembles Urocanate Hydratase from Geobacillus kaustophilus (GkUH) (open conformation), while monomer C resembles Urocanate Hydratase from Pseudomonas putida (PpUH) and Urocanate Hydratase from Bacillus subtilis (BsUH) (closed conformations). We use the structure of TcUH to make considerations about 3 non-deleterious and 2 deleterious mutations found in human UHs: non-deleterious mutations could be accommodated without large displacements or interaction interruptions, whereas deleterious mutations in one case might disrupt an α-helix (as previously suggested) and in the other case, besides disrupting the enzyme interaction with the substrate, might interfere with interdomain movement.

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state.

Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD+ as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD+ functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD+ with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD+-mediated "gate keeper" function involving NAD+/NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD+ in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD+-mediated "gate keeper" and substrate tunnel may assist in the exploration the NAD+ function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis.

Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis.

Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric forms of baFabI in the apo state and in complex with NAD+ and with NAD+-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix η7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of baFabI is required for cofactor binding and catalytic activity.

Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

Energy conversion, redox catalysis and generation of reactive oxygen species by respiratory complex I.

Complex I (NADH:ubiquinone oxidoreductase) is critical for respiration in mammalian mitochondria. It oxidizes NADH produced by the Krebs' tricarboxylic acid cycle and ß-oxidation of fatty acids, reduces ubiquinone, and transports protons to contribute to the proton-motive force across the inner membrane. Complex I is also a significant contributor to cellular oxidative stress. In complex I, NADH oxidation by a flavin mononucleotide, followed by intramolecular electron transfer along a chain of iron-sulfur clusters, delivers electrons and energy to bound ubiquinone. Either at cluster N2 (the terminal cluster in the chain) or upon the binding/reduction/dissociation of ubiquinone/ubiquinol, energy from the redox process is captured to initiate long-range energy transfer through the complex and drive proton translocation. This review focuses on current knowledge of how the redox reaction and proton transfer are coupled, with particular emphasis on the formation and role of semiquinone intermediates in both energy transduction and reactive oxygen species production. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Oxidation of NADH and ROS production by respiratory complex I.

Kinetic characteristics of the proton-pumping NADH:quinone reductases (respiratory complexes I) are reviewed. Unsolved problems of the redox-linked proton translocation activities are outlined. The parameters of complex I-mediated superoxide/hydrogen peroxide generation are summarized, and the physiological significance of mitochondrial ROS production is discussed. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Infrared spectroscopic studies on reaction induced conformational changes in the NADH ubiquinone oxidoreductase (complex I).

Redox-dependent conformational changes are currently discussed to be a crucial part of the reaction mechanism of the respiratory complex I. Specialized difference Fourier transform infrared techniques allow the detection of side-chain movements and minute secondary structure changes. For complex I, (1)H/(2)H exchange kinetics of the amide modes revealed a better accessibility of the backbone in the presence of NADH and quinone. Interestingly, the presence of phospholipids, that is crucial for the catalytic activity of the isolated enzyme complex, changes the overall conformation. When comparing complex I samples from different species, very similar electrochemically induced FTIR difference spectra and very similar rearrangements are reported. Finally, the information obtained with variants and from Zn(2+) inhibited samples for the conformational reorganization of complex I upon electron transfer are discussed in this review. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.

α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress.

Magnetic nano-Fe3O4-supported 1-benzyl-1,4-dihydronicotinamide (BNAH): synthesis and application in the catalytic reduction of α,ß-epoxy ketones.

A novel magnetically recoverable organic hydride compound was successfully constructed by using silica-coated magnetic nanoparticles as a support. An as-prepared magnetic organic hydride compound, BNAH (1-benzyl-1,4-dihydronicotinamide), showed efficient activity in the catalytic reduction of α,ß-epoxy ketones. After reaction, the magnetic nanoparticle-supported BNAH can be separated by simple magnetic separation which made the separation of the product easier.

Signal-enhanced electrochemiluminescence immunosensor based on synergistic catalysis of nicotinamide adenine dinucleotide hydride and silver nanoparticles.

A new metal-organic nanocomposite with synergistic catalysis function was prepared and developed to construct an electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of tumor biomarker CA125. Silver nanoparticles (AgNPs) and nicotinamide adenine dinucleotide hydride (NADH) that can participate and catalyze the ECL reaction of Ru(bpy)(3)(2+) were employed as the metal component and the organic component to synthesize the metal-organic nanocomposite of NADH-AgNPs (NA). The novel ECL immunosensor was assembled via Ru(bpy)(3)(2+)-doped silica nanoparticles (Ru-SiO(2)) modified electrode with the NA as immune labels. First, the chitosan-suspended Ru-SiO(2) nanoparticles were cast on the gold electrode surface to immobilize the ECL probes of Ru(bpy)(3)(2+) and link gold nanoparticles. Then, the primary antibodies were loaded onto the modified electrode via the gold sulfhydryl covalent binding. After immunobinding the analytes of antigen, NA-attached secondary antibodies could be captured as a sandwich type on the electrode. Finally, based on the circularly synergistic catalysis by the silver and NADH for the solid-phase ECL of Ru(bpy)(3)(2+), the proposed immunosensor sensed the concentration of antigen. The synergistic ECL catalysis of metal-organic nanocomposite amplified response signal and pushed the detection limit down to 0.03 U ml(-1), which initiated a new ECL labeling field and has great significance for ECL immunoassays.