Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates.

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2 Here, using electron paramagnetic resonance (EPR), Mössbauer, and UV-visible spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C-F Bond Scission.

Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyr•) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C-H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAOV previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAOV, Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Šresolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyr•-Cys) or Cu(II)-(F-Tyr•-Cys). These findings illustrate a previously unobserved C-F/C-Cl bond cleavage in biology mediated by a mononuclear copper center.

Cleavage of a carbon-fluorine bond by an engineered cysteine dioxygenase.

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93-Tyr157 cross-linked cofactor. Here, we investigated this Cys-Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 19F NMR, these data indicated that the enzyme could catalyze oxidative C-F or C-Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys-Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon-halogen bond activation.

Ultrastable silver nanoparticles.

Noble-metal nanoparticles have had a substantial impact across a diverse range of fields, including catalysis, sensing, photochemistry, optoelectronics, energy conversion and medicine. Although silver has very desirable physical properties, good relative abundance and low cost, gold nanoparticles have been widely favoured owing to their proved stability and ease of use. Unlike gold, silver is notorious for its susceptibility to oxidation (tarnishing), which has limited the development of important silver-based nanomaterials. Despite two decades of synthetic efforts, silver nanoparticles that are inert or have long-term stability remain unrealized. Here we report a simple synthetic protocol for producing ultrastable silver nanoparticles, yielding a single-sized molecular product in very large quantities with quantitative yield and without the need for size sorting. The stability, purity and yield are substantially better than those for other metal nanoparticles, including gold, owing to an effective stabilization mechanism. The particular size and stoichiometry of the product were found to be insensitive to variations in synthesis parameters. The chemical stability and structural, electronic and optical properties can be understood using first-principles electronic structure theory based on an experimental single-crystal X-ray structure. Although several structures have been determined for protected gold nanoclusters, none has been reported so far for silver nanoparticles. The total structure of a thiolate-protected silver nanocluster reported here uncovers the unique structure of the silver thiolate protecting layer, consisting of Ag2S5 capping structures. The outstanding stability of the nanoparticle is attributed to a closed-shell 18-electron configuration with a large energy gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital, an ultrastable 32-silver-atom excavated-dodecahedral core consisting of a hollow 12-silver-atom icosahedron encapsulated by a 20-silver-atom dodecahedron, and the choice of protective coordinating ligands. The straightforward synthesis of large quantities of pure molecular product promises to make this class of materials widely available for further research and technology development.

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening.

Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1H NMR, 13C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.

Cofactor Biogenesis in Cysteamine Dioxygenase: C-F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine.

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F2 -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and 19 F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink.

Hormone glucagon: electrooxidation and determination at carbon nanotubes.

The oxidation of glucagon, which is one of the key hormones in glucose homeostasis, was studied at electrodes modified with carbon nanotubes (CNT) that were dispersed in a polysaccharide adhesive chitosan (CHIT). Such electrodes displayed improved resistance to fouling, which allowed for the investigation of both the electrolysis/mass spectrometry and electroanalysis of glucagon. The off-line electrospray ionization and tandem mass spectrometric analyses showed that the -4 Da mass change to glucagon upon electrolysis at CNT was due to the electrooxidation of its tryptophan (W25) and dityrosine (Y10, Y13) residues. The methionine residue of glucagon did not contribute to its oxidation. The amperometric determination of glucagon yielded the limit of detection equal to ∼20 nM (E = 0.800 V, pH 7.40, S/N = 3), sensitivity of 0.46 A M(-1) cm(-2), linear dynamic range up to 2.0 µM (R(2) = 0.998), response time <5 s, and good signal stability. Free tryptophan and tyrosine yielded comparable analytical figures of merit. The direct amperometric determination of unlabeled glucagon at CHIT-CNT electrodes is the first example of a rapid alternative to the complex analytical assays of this peptide.

M3Ag17(SPh)12 Nanoparticles and Their Structure Prediction.

Although silver nanoparticles are of great fundamental and practical interest, only one structure has been determined thus far: M4Ag44(SPh)30, where M is a monocation, and SPh is an aromatic thiolate ligand. This is in part due to the fact that no other molecular silver nanoparticles have been synthesized with aromatic thiolate ligands. Here we report the synthesis of M3Ag17(4-tert-butylbenzene-thiol)12, which has good stability and an unusual optical spectrum. We also present a rational strategy for predicting the structure of this molecule. First-principles calculations support the structural model, predict a HOMO-LUMO energy gap of 1.77 eV, and predict a new "monomer mount" capping motif, Ag(SR)3, for Ag nanoparticles. The calculated optical absorption spectrum is in good correspondence with the measured spectrum. Heteroatom substitution was also used as a structural probe. First-principles calculations based on the structural model predicted a strong preference for a single Au atom substitution in agreement with experiment.

Dietary Supplementation with 23-Hydroxy Ursolic Acid Reduces the Severity and Incidence of Acute Experimental Autoimmune Encephalomyelitis (EAE) in a Murine Model of Multiple Sclerosis.

23-Hydroxy ursolic acid (23-OH UA) is a potent atheroprotective and anti-obesogenic phytochemical, with anti-inflammatory and inflammation-resolving properties. In this study, we examined whether dietary 23-OH UA protects mice against the acute onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Female C57BL/6 mice were fed either a defined low-calorie maintenance diet (MD) or an MD supplemented with 0.2% wgt/wgt 23-OH UA for 5 weeks prior to actively inducing EAE and during the 30 days post-immunization. We observed no difference in the onset of EAE between the groups of mice, but ataxia and EAE disease severity were suppressed by 52% and 48%, respectively, and disease incidence was reduced by over 49% in mice that received 23-OH UA in their diet. Furthermore, disease-associated weight loss was strikingly ameliorated in 23-OH UA-fed mice. ELISPOT analysis showed no significant differences in frequencies of T cells producing IL-17 or IFN-γ between 23-OH UA-fed mice and control mice, suggesting that 23-OH UA does not appear to regulate peripheral T cell responses. In summary, our findings in EAE mice strongly suggest that dietary 23-OH UA may represent an effective oral adjunct therapy for the prevention and treatment of relapsing-remitting MS.

Targeting APEX2 to the mRNA encoding fatty acid synthase ß in yeast identifies interacting proteins that control its abundance in the cell cycle.

Profiling the repertoire of proteins associated with a given mRNA during the cell cycle is unstudied. Furthermore, it is easier to ask and answer what mRNAs a specific protein might bind to than the other way around. Here, we implemented an RNA-centric proximity labeling technology at different points in the cell cycle in highly synchronous yeast cultures. To understand how the abundance of FAS1, encoding fatty acid synthase, peaks late in the cell cycle, we identified proteins that interact with the FAS1 transcript in a cell cycle-dependent manner. We used dCas13d-APEX2 fusions to target FAS1 and label nearby proteins, which were then identified by mass spectrometry. The glycolytic enzyme Tdh3p, a known RNA-binding protein, interacted with the FAS1 mRNA, and it was necessary for the periodic abundance of Fas1p in the cell cycle. These results point to unexpected connections between major metabolic pathways. They also underscore the role of mRNA-protein interactions for gene expression during cell division.

Mass spectrometric identification of silver nanoparticles: the case of Ag32(SG)19.

Mass spectrometry has played a key role in identifying the members of a series of gold clusters, which has enabled the development of magic-number cluster theory. The successes of the gold cluster system have yet to be repeated in another metal cluster system, however. Silver clusters in particular have proven to be challenging due to their relative instability compared with gold clusters. Using the well-characterized gold nanocluster, Au(25)(SG)(18), we present optimized electrospray ionization mass spectrometry (ESI-MS) instrumental parameters for the maximal transmission of the intact cluster. Parameters shown to have the largest effect on intact cluster transmission/detection include trap and transfer collision energy, source temperature, and cone gas flow rate. Herein we describe a general strategy to acquire mass spectra of fragile metal clusters with reliable mass assignments. By also optimizing sample solution conditions, high-quality ESI mass spectra of a prototypical silver:glutathione (Ag:SG) cluster were obtained without significant fragmentation. By using gentle conditions and solution conditions designed to stabilize the clusters, fragmentation was dramatically reduced and mass spectra with isotopic resolution were measured. Using this strategy, we have made the first formula assignment for a ligand-protected Ag cluster of Ag(32)(SG)(19).

Analysis of Histone Modifications from Tryptic Peptides of Deuteroacetylated Isoforms.

The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.

Screening method for isopeptides from small ubiquitin-related modifier-conjugated proteins by ion mobility mass spectrometry.

Posttranslational modification by the small ubiquitin-related modifier (SUMO) is a highly regulated modification, which is often restricted to very specific cellular events. A number of analytical strategies for identification of SUMOylated proteins have been previously reported in the literature. A new screening method for SUMOylated peptides based on ion mobility mass spectrometry is presented. Using poly-SUMO2 as a model system, a two-enzyme trypsin/chymotrypsin digestion was performed to reduce the size of the isopeptide conjugated to the substrate lysine residue. Traveling wave ion mobility mass spectrometry was used to screen for peptides containing the QQQTGG isopeptide tag from SUMO, which increases the mass and size of the peptide by 618 Da. This increase in mass along with solution conditions to promote higher charge states allows the isopeptides to be separated from the typically smaller and lesser charged linear peptides. On the basis of these findings, this method can be used as a quick and easy screening method for identifying possible SUMO isopeptides.

Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins.

Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.

The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.

BACKGROUND: Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown. RESULTS: The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1. CONCLUSIONS: Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.

The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.

Biocatalytic Carbon-Hydrogen and Carbon-Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme.

LmbB2 is a peroxygenase-like enzyme that hydroxylates L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize L-tyrosine analogs with ring-deactivated substituents such as 3-nitro-, fluoro-, chloro-, iodo-L-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C-H bond. The most interesting observation of this study was obtained with 3-fluoro-L-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations, i.e., oxidative C-H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-L-tyrosine and oxygenation at C3 concomitant with a carbon-fluorine bond cleavage to yield DOPA and fluoride. An iron protein-mediated hydroxylation on both C-H and C-F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C-H/C-X bond (X = halogen) cleavage. Further 18O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide, and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C-F bond cleavage. Instead, the C-F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron-hydroperoxo species. Based on the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C-H/C-F bond. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.

Chemical synthesis of 7α-hydroxypregnenolone, a neuroactive steroid that stimulates locomotor activity.

7α-Hydroxypregnenolone is an endogenous neuroactive steroid that stimulates locomotor activity. A synthesis of 7α-hydroxypregnenolone from pregnenolone, which takes advantage of an orthogonal protecting group strategy, is described. In detail, the C7-position was oxidized with CrO3 and 3,5-dimethylpyrazole to yield a 7-keto steroid intermediate. The resulting 7-ketone was stereoselectively reduced to the 7α-hydroxy group with lithium tri-sec-butylborohydride. In contrast, reduction of the same 7-ketone intermediate with NaBH4 resulted in primarily the 7ß-hydroxy epimer. Furthermore, in an alternative route to the target compound, the 7α-hydroxy group was successfully incorporated by direct C-H allylic benzoyloxylation of pregnenolone-3-acetate with CuBr and tert-butyl peroxybenzoate followed by saponification. The disclosed syntheses to 7-oxygenated steroids are amenable to potentially obtain other biologically active sterols and steroids.