Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5\'-deoxy-5\'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including \"negative catalysis\", a paradigm for AdoCbl-dependent mutases.

This study explored the antitubercular properties of fucoxanthin, a marine carotenoid, against clinical isolates of Mycobacterium tuberculosis (Mtb). Two vital enzymes involved in Mtb cell wall biosynthesis, UDP-galactopyranose mutase (UGM) and arylamine-N-acetyltransferase (TBNAT), were selected as drug targets to reveal the mechanism underlying the antitubercular effect of fucoxanthin. The obtained results showed that fucoxanthin showed a clear bacteriostatic action against the all Mtb strains tested, with minimum inhibitory concentrations (MIC) ranging from 2.8 to 4.1 µM, along with a good degree of selectivity index (ranging from 6.1 to 8.9) based on cellular toxicity evaluation compared with standard drug isoniazid (INH). The potent inhibitory actions of fucoxanthin and standard uridine-5\'-diphosphate against UGM were recorded to be 98.2% and 99.2%, respectively. TBNAT was potently inactivated by fucoxanthin (half maximal inhibitory concentration (IC50) = 4.8 µM; 99.1% inhibition) as compared to INH (IC50 = 5.9 µM; 97.4% inhibition). Further, molecular docking approaches were achieved to endorse and rationalize the biological findings along with envisaging structure-activity relationships. Since the clinical evidence of the last decade has confirmed the correlation between bacterial infections and autoimmune diseases, in this study we have discussed the linkage between infection with Mtb and autoimmune diseases based on previous clinical observations and animal studies. In conclusion, we propose that fucoxanthin could demonstrate great therapeutic value for the treatment of tuberculosis by acting on multiple targets through a bacteriostatic effect as well as by inhibiting UGM and TBNAT. Such outcomes may lead to avoiding or decreasing the susceptibility to autoimmune diseases associated with Mtb infection in a genetically susceptible host.

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

In bacterial and archaeal purine biosynthetic pathways, sixth step involves utilization of enzyme PurE, catalyzing the translation of aminoimidazole ribonucleotide to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) with carbon dioxide. The formation of CAIR takes place through an unstable intermediate N5-CAIR, played by two enzymes-N5-CAIR synthetase (PurK) and N5-CAIR mutase (PurE) that further catalyzes the reaction of N5-CAIR to CAIR. In this study, N5-CAIR mutase (PH0320) from Pyrococcus horikoshii OT3 (PurE) was considered. The three-dimensional structure of Pyrococcus horikoshii OT3 was modeled based on the structure of PurE from Escherichia coli. The modeled structure was subjected to molecular dynamics simulation up to 100 ns, and least energy structure from the simulation was subjected to virtual screening and induced fit docking to identify the best potent leads. A total of five best antagonists were identified based on their affinity and mode of binding leading with conserved residues Ser18, Ser20, Asp21, Ser45, Ala46, His47, Arg48, Ala72, Gly73, Ala75, and His77 promotes the activity of Ph-N5-CAIR mutase. In addition to molecular dynamics, absorption, digestion, metabolism, and excretion properties, binding free energy and density functional theory calculations of compounds were carried out. Based on analyses, compound from National Cancer Institute (NCI) database, NCI_826 was adjudged as the best potent lead molecule and could be suggested as the suitable inhibitor of N5-CAIR mutase.

Uridine 5\'-diphosphate-5-fluorogalactopyranose (UDP-5F-Galp, 7) was synthesized, and its effect on UDP-Galp mutase (UGM) was investigated. UGM facilitated the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but no 11 was detected. (19)F NMR and trapping experiments demonstrated that the reaction involves the initial formation of a substrate-cofactor adduct followed by decomposition of the resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate (10). The results support the current mechanistic proposal for UGM and suggest new directions for designing mechanism-based inhibitors.

The enzyme UDP-Galactopyranose Mutase (UGM) catalyses the conversion of galactopyranose into galactofuranose. It is known to be critical for the survival and proliferation of several pathogenic agents, both prokaryotic and eukaryotic. Among them is Trypanosoma cruzi, the parasite responsible for Chagas\' disease. Since the enzyme is not present in mammals, it appears as a promising target for the design of drugs to treat this illness. A precise knowledge of the mechanism of the catalysed reaction would be crucial to assist in such design. In this article we present a detailed study of all the putative steps of the mechanism. The study is based on QM/MM free energy calculations along properly selected reaction coordinates, and on the analysis of the main structural changes and interactions taking place at every step. The results are discussed in connection with the experimental evidence and previous theoretical studies.

The function of the active-site residues of oxidosqualene cyclases (OSCs) has been presumed mainly in light of the product distribution; however, not much research has been performed into the enzymatic activity of mutated OSCs. β-Amyrin, which is widely found in the plant kingdom, is classified as an OSC; mutational studies on β-amyrin cyclase are very limited. Six site-specific mutations targeted at the Phe728 residue of Euphorbia tirucalli β-amyrin synthase (EtAS) were constructed to inspect the function of this aromatic residue. We developed a simple method to evaluate the in vivo enzymatic activity; the expression levels of EtASs and the quantities of the cyclic triterpenes produced were determined by use of western blot and GC analyses, respectively. Measurement of the relative in vivo activity of the mutants versus that of the wild-type enzyme showed that the Ala, Met, His, and Trp variants had significantly decreased activity, but that the Tyr mutant had a high activity, which was nearly the same as that of the wild-type enzyme. In contrast to Tyr, Ala and Met possess no π-electrons; thus, the role of Phe728 is to stabilize the cationic intermediates, resulting in facilitation of the ring-expansion processes, especially by stabilizing the secondary cations. The decreased activity of the Trp mutant is ascribed to the introduction of a large steric bulk, leading to looser binding of oxidosqualene in the Trp variant. The His mutant afforded germanicol as the main product, indicating that the Phe residue is located near the D/E-ring-formation site. Changes in the steric bulk gave some cationic intermediates, resulting in the formation of 13 cyclic triterpenes, including an unnatural triterpene, (17E)-dammara-17(20),24-dien-3β-ol, and isoursenol, which has rarely been found in nature. In this study, we provide the first experimental evidence that cation-π interactions play a key role in the catalytic action of OSCs.

The Taxus canadensis phenylalanine aminomutase (TcPAM) catalyze the isomerization of (S)-α-phenylalanine to the (R)-β-isomer. The active site of TcPAM contains the signature 5-methylene-3,5-dihydroimidazol-4-one (MIO) prosthesis, observed in the ammonia lyase class of enzymes. Up to now, there are two plausible mechanisms for these MIO-dependent enzymes, i.e., the amino-MIO adduct mechanism and the Friedel-Crafts-type reaction mechanism. In response to this mechanistic uncertainty, the phenylalanine aminomutase mechanism was investigated by using density functional methods. The calculation results indicate that: (1) the reaction prefers the amino-MIO adduct mechanism where the 2,3-amine shift process contains six elementary steps; (2) the ammonia elimination step proceeds through an E2 mechanism; (3) a single C1Cα bond rotation of 180° in the cinnamate skeleton occurs in the active site prior to the rebinding of NH2 group to the cinnamate. This can be used to explain the stereochemistry of the TcPAM reaction product which is contrary to those of the PaPAM and SgTAM enzymes. Based on these calculations, the roles of important residues in the active site were also elucidated.

Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.