Metabolomics analysis of urine from patients with alcohol-associated liver disease reveals dysregulated caffeine metabolism.

Excess alcohol intake causes millions of deaths annually worldwide. Asymptomatic early-stage, alcohol-associated liver disease (ALD) is easily overlooked, and ALD is usually only diagnosed in more advanced stages. We explored the possibility of using polar urine metabolites as biomarkers of ALD for early-stage diagnosis and functional assessment of disease severity by quantifying the abundance of polar metabolites in the urine samples of healthy controls (n = 18), patients with mild or moderate liver injury (n = 21), and patients with severe alcohol-associated hepatitis (n = 25). The polar metabolites in human urine were first analyzed by untargeted metabolomics, showing that 209 urine metabolites are significantly changed in patients, and 17 of these are highly correlated with patients' model for end-stage liver disease (MELD) score. Pathway enrichment analysis reveals that the caffeine metabolic pathway is the most affected in ALD. We then developed a targeted metabolomics method and measured the concentration of caffeine and its metabolites in urine using internal and external standard calibration, respectively. The described method can quantify caffeine and its 14 metabolites in 35 min. The results of targeted metabolomics analysis agree with the results of untargeted metabolomics, showing that 13 caffeine metabolites are significantly decreased in patients. In particular, the concentrations of 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil are markedly decreased with increased disease severity. We suggest that these three metabolites could serve as functional biomarkers for differentiating early-stage ALD from more advanced liver injury.NEW & NOTEWORTHY Our study using both untargeted and targeted metabolomics reveals the caffeine metabolic pathway is dysregulated in ALD. Three caffeine metabolites, 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil, can differentiate the severity of early-stage ALD.

The Pseudouridine Synthases Proceed through a Glycal Intermediate.

The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

Metabolomic analysis of the effects of polychlorinated biphenyls in nonalcoholic fatty liver disease.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants and have been associated with abnormal liver enzymes and suspected nonalcoholic fatty liver disease (NAFLD), obesity, and the metabolic syndrome in epidemiological studies. In epidemiological surveys of human PCB exposure, PCB 153 has the highest serum levels among PCB congeners. To determine the hepatic effects of PCB 153 in mice, C57BL/6J mice were fed either a control diet (CD) or a high fat diet (HFD) for 12 weeks, with or without PCB 153 coexposure. The metabolite extracts from mouse livers were analyzed using linear trap quadrupole-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR MS) via direct infusion nanoelectrospray ionization (DI-nESI) mass spectrometry. The metabolomics analysis indicated no difference in the metabolic profile between mice fed the control diet with PCB 153 exposure (CD+PCB 153) and mice fed the control diet (CD) without PCB 153 exposure. However, compared with CD group, levels of 10 metabolites were increased and 15 metabolites were reduced in mice fed HFD. Moreover, compared to CD+PCB 153 group, the abundances of 6 metabolites were increased and 18 metabolites were decreased in the mice fed high fat diet with PCB 153 exposure (HFD+PCB 153). Compared with HFD group, the abundances of 2 metabolites were increased and of 12 metabolites were reduced in HFD+PCB 153 group. These observations agree with the histological results and indicate that the metabolic effects of PCB 153 were highly dependent on macronutrient interactions with HFD. Antioxidant depletion is likely to be an important consequence of this interaction, as this metabolic disturbance has previously been implicated in obesity and NAFLD.

The handling of the mechanistic probe 5-fluorouridine by the pseudouridine synthase TruA and its consistency with the handling of the same probe by the pseudouridine synthases TruB and RluA.

RNA containing 5-fluorouridine (F(5)U) had previously been used to examine the mechanism of the pseudouridine synthase TruA, formerly known as pseudouridine synthase I [Gu et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]. From that work, it was reasonably concluded that the pseudouridine synthases proceed via a mechanism involving a Michael addition by an active site aspartic acid residue to the pyrimidine ring of uridine or F(5)U. Those conclusions rested on the assumption that the hydrate of F(5)U was obtained after digestion of the product RNA and that hydration resulted from hydrolysis of the ester intermediate between the aspartic acid residue and F(5)U. As reported here, (18)O labeling definitively demonstrates that ester hydrolysis does not give rise to the observed hydrated product and that digestion generates not the expected mononucleoside product but rather a dinucleotide between a hydrated isomer of F(5)U and the following nucleoside in RNA. The discovery that digestion products are dinucleotides accounts for the previously puzzling differences in the isolated products obtained following the action of the pseudouridine synthases TruB and RluA on F(5)U in RNA.

The products of 5-fluorouridine by the action of the pseudouridine synthase TruB disfavor one mechanism and suggest another.

The pseudouridine synthase TruB handles 5-fluorouridine in RNA as a substrate, converting it into two isomeric hydrated products. Unexpectedly, the two products differ not in the hydrated pyrimidine ring but in the pentose ring, which is epimerized to arabinose in the minor product. This inversion of stereochemistry at C2' suggests that pseudouridine generation may proceed by a mechanism involving a glycal intermediate or that the previously proposed mechanism involving an acylal intermediate operates but with an added reaction manifold for 5-fluorouridine versus uridine. The arabino product strongly disfavors a mechanism involving a Michael addition to the pyrimidine ring.

Se-ing into selenocysteine biosynthesis.

A cocrystal structure of the enzyme that synthesizes selenocysteine reveals the elegantly simple recognition mechanism for the tRNA molecule for this '21st amino acid'. The structure resolves some mechanistic questions and allows for comparison of the tRNA-dependent synthesis of cysteine and selenocysteine.

Unexpected linear ion trap collision-induced dissociation and Fourier transform ion cyclotron resonance infrared multi-photon dissociation fragmentation of a hydrated C-glycoside of 5-fluorouridine formed by the action of the pseudouridine synthases RluA and TruB.

As part of the investigation of the pseudouridine synthases, 5-fluorouridine in RNA was employed as a mechanistic probe. The hydrated, rearranged product of 5-fluorouridine was isolated as part of a dinucleotide and found to undergo unusual fragmentation during mass spectrometry, with the facile loss of HNCO from the product pyrimidine ring favored over phosphodiester bond rupture. Although the loss of HNCO from uridine and pseudouridine is well established, the pericyclic process leading to their fragmentation cannot operate with the saturated pyrimidine ring in the product of 5-fluorouridine. Based on the MS(n) results and calculations reported here, a new mechanism relying on the peculiar disposition of the functional groups of the product pyrimidine ring is proposed to account for the unusually facile fragmentation.

Kinetic Isotope Effect Studies to Elucidate the Reaction Mechanism of RNA-Modifying Enzymes.

The synthesis of specifically deuterated uridine, its incorporation into an RNA oligonucleotide substrate, and the use of the labeled substrate to determine the deuterium kinetic isotope effect for the reaction catalyzed by the pseudouridine synthases (enzymes that isomerize uridine to pseudouridine in RNA) are described. Both enzymes-TruB and RluA-display a primary kinetic isotope effect, which indicates the formation of a glycal intermediate in the ribose ring during turnover. Although the details of the protocols are specific to these two enzymes, the general methodology is readily adaptable to the synthesis and incorporation of other labeled nucleosides into any RNA molecule by in vitro transcription.

Direct evidence for enzyme persulfide and disulfide intermediates during 4-thiouridine biosynthesis.

Two proposed mechanisms for 4-thiouridine generation share key cysteine persulfide and disulfide intermediates, and indirect evidence of their existence has been previously reported; chemical trapping and mass spectrometry have now provided direct and definitive evidence of these key intermediates.

Characterization of the catalytic disulfide bond in E. coli 4-thiouridine synthetase to elucidate its functional quaternary structure.

4-Thiouridine at position 8 in prokaryotic tRNA serves as a photosensor for near-UV light, and the posttranscriptional conversion of uridine to 4-thiouridine is catalyzed by the 4-thiouridine synthetases (s(4) US, also named ThiI), which fall into two classes that differ in the presence of a C-terminal rhodanese homology domain. A cysteine residue in this domain first bears a persulfide group and then forms a disulfide bond with a cysteine residue that is conserved in both classes of s(4) US. Recent crystal structures suggest that s(4) US dimerizes in the presence of RNA substrate with domains from each subunit contributing to the binding and reaction of one RNA molecule, which raises the question of whether the catalytic disulfide bond in the longer class of s(4) US is formed within or between subunits. The E. coli enzyme is the best-characterized member of the longer class of s(4) US, and it was examined after quantitative installation of the disulfide bond during a single catalytic turnover. Gel electrophoresis and proteolysis/MALDI-MS results strongly imply that the disulfide bond forms within a single subunit, which provides a vital constraint for the structural modeling of the class of s(4) US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.

Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

The pseudouridine synthase TruB is responsible for the universally conserved post-transcriptional modification of residue 55 of elongator tRNAs. In addition to the active site, the "thumb", a peripheral domain unique to the TruB family of enzymes, makes extensive interactions with the substrate. To coordinate RNA binding and release with catalysis, the thumb may be able to sense progress of the reaction in the active site. To establish whether there is a structural correlate of communication between the active site and the RNA-sequestering thumb, we have solved the structure of a catalytically inactive point mutant of TruB in complex with a substrate RNA, and compared it to the previously determined structure of an active TruB bound to a reaction product. Superposition of the two structures shows that they are extremely similar, except in the active site and, intriguingly, in the relative position of the thumb. Because the two structures were solved using isomorphous crystals, and because the thumb is very well ordered in both structures, the displacement of the thumb we observe likely reflects preferential propagation of active site perturbations to this RNA-binding domain. One of the interactions between the active site and the thumb involves an active site residue whose hydrogen-bonding status changes during the reaction. This may allow the peripheral RNA-binding domain to monitor progress of the pseudouridylation reaction.

A paradigm for biological sulfur transfers via persulfide groups: a persulfide-disulfide-thiol cycle in 4-thiouridine biosynthesis.

In support of the key features of sulfur transfer in the proposed mechanisms of 4-thiouridine generation, the enzyme ThiI can turn over only once in the absence of reductants of disulfide bonds, and Cys-456 of ThiI receives the sulfur transferred from the persulfide group of the sulfurtransferase IscS.

Trafficking in persulfides: delivering sulfur in biosynthetic pathways.

The presence of sulfur in cofactors has been appreciated for over a century, but the trafficking and delivery of sulfur to cofactors and nucleosides is still not fully understood. In the last decade, great strides have been made toward understanding those processes and the enzymes that conduct them, including cysteine desulfurases and rhodanese homology domain proteins. The persulfide group (R-S-SH) predominantly serves as the sulfur donor, and sulfur incorporation pathways share enzymes to a remarkable degree. Mechanisms for the use of persulfide groups are illustrated with the relatively simple case of 4-thiourdine generation, and further possibilities are illuminated by the 2-thiouridine and cofactor biosynthetic systems. The rationale and ramifications of sharing enzymes between sulfur incorporation pathways are discussed, including implications for interpreting genetic or genomic data that indicate a role for a sulfur transfer protein in a particular biological process.

Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine.

The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the psi synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of psi synthases and an enzyme that makes an adduct with f5U in RNA, the behavior of RluA toward RNA containing f5U was examined. Stem-loop RNAs are shown to be good substrates for RluA. Heat denaturation of the adduct between RluA and RNA containing f5U produces a hydrated nucleoside product, and labeling studies show that hydration does not occur by ester hydrolysis. These results are interpreted in light of a consistent mechanistic scheme for the handling of f5U by psi synthases.

Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure.

RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adopting its canonical U turn conformation, the anticodon loop folds into a new structure with a reverse-Hoogsteen base pair and three flipped-out nucleotides. Sequence conservation, the cocrystal structure, and the results of structure-guided mutagenesis suggest that RluA recognizes its substrates indirectly by probing RNA loops for their ability to adopt the reorganized fold. The planar, cationic side chain of an arginine intercalates between the reverse-Hoogsteen base pair and the bottom pair of the anticodon stem, flipping the nucleotide to be modified into the active site of RluA. Sequence and structural comparisons suggest that pseudouridine synthases of the RluA, RsuA, and TruA families employ an equivalent arginine for base flipping.

The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB.

All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.

Not all pseudouridine synthases are potently inhibited by RNA containing 5-fluorouridine.

RNA containing 5-fluorouridine has been assumed to inhibit strongly or irreversibly the pseudouridine synthases that act on the RNA. RNA transcripts containing 5-fluorouridine in place of uridine have, therefore, been added to reconstituted systems in order to investigate the importance of particular pseudouridine residues in a given RNA by inactivating the pseudouridine synthase responsible for their generation. In sharp contradiction to the assumption of universal inhibition of pseudouridine synthases by RNA containing 5-fluorouridine, the Escherichia coli pseudouridine synthase TruB, which has physiologically critical eukaryotic homologs, is not inhibited by such RNA. Instead, the RNA containing 5-fluorouridine was handled as a substrate by TruB. The E. coli pseudouridine synthase RluA, on the other hand, forms a covalent complex and is inhibited stoichiometrically by RNA containing 5-fluorouridine. We offer a hypothesis for this disparate behavior and urge caution in interpreting results from reconstitution experiments in which RNA containing 5-fluorouridine is assumed to inhibit a pseudouridine synthase, as normal function may result from a failure to inactivate the targeted enzyme rather than from the absence of nonessential pseudouridine residues.

The pseudouridine synthases: revisiting a mechanism that seemed settled.

RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.