Eugene G. Mueller

Critical Aspartic Acid Residues in Pseudouridine Synthases

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry 37, 344–351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

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.

Chips off the old block

A lack of sequence similarity made it uncertain whether the four families of pseudouridine synthases arose by convergent or divergent evolution. The structures of three of these enzymes reveal that they are homologs, but a paucity of obvious catalytic residues in the active site reopens the question of a mechanism that seemed settled.

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.

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.

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.

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 (s4US, 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 s4US. Recent crystal structures suggest that s4US 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 s4US is formed within or between subunits. The E. coli enzyme is the best‐characterized member of the longer class of s4US, 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 s4US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.

A paradigm for biological sulfur transfers via persulfide groups: a persulfide???disulfide???thiol cycle in 4-thiouridine biosynthesisElectronic supplementary information (ESI) available: complete experimental details. See http://www.rsc.org/suppdata/cc/b2/b208626c/

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.

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.

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.