Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites

Soga T.

Capillary electrophoresis mass spectrometry (CE-MS) is now regarded as a powerful tool for the comprehensive analysis of low-molecular-weight charged metabolites. Over the past decades, several CE-MS methods have been established and made significant contributions to metabolomics research in various areas. This review describes the general strategies and principles of the CE-MS-based metabolomics analyses, including sample preparation, CE separation modes, interfacing techniques, and mass spectrometers. Furthermore, new techniques to increase throughput ability in CE-MS for metabolomics are highlighted. Finally, general conclusions and perspectives are provided.

Frohnmeyer H., Elling L.

Nucleotide sugars play an elementary role in nature as building blocks of glycans, polysaccharides, and glycoconjugates used in the pharmaceutical, cosmetics, and food industries. As substrates of Leloir-glycosyltransferases, nucleotide sugars are essential for chemoenzymatic in vitro syntheses. However, high costs and the limited availability of nucleotide sugars prevent applications of biocatalytic cascades on a large industrial scale. Therefore, the focus is increasingly on nucleotide sugar synthesis strategies to make significant application processes feasible. The chemical synthesis of nucleotide sugars and their derivatives is well established, but the yields of these processes are usually low. Enzyme catalysis offers a suitable alternative here, and in the last 30 years, many synthesis routes for nucleotide sugars have been discovered and used for production. However, many of the published procedures shy away from assessing the practicability of their processes. With this review, we give an insight into the development of the (chemo)enzymatic nucleotide sugar synthesis pathways of the last years and present an assessment of critical process parameters such as total turnover number (TTN), space-time yield (STY), and enzyme loading.

Winston M.S., Poirier M., Liu Z., Peng F., Humphrey G.R., McIntosh J.A., Reibarkh M., Wang F., Guetschow E.D., Castro S., Hoyt E., Lamberto D.J., Sirk K.

Li S., Zhang Z., Liu F., Yuan B., Liu T., Feng Y.

Bhinderwala F., E. Roth H., Noel H., Feng D., Powers R.

The reliability and robustness of metabolite assignments in 1H NMR is complicated by numerous factors including variations in temperature, pH, buffer choice, ionic strength, and mixture composition that led to peak overlap and spectral crowding. As sample conditions fluctuate, peak drift and line broadening further complicate peak deconvolution and subsequent chemical assignment. We present a collection of 1D 1H NMR spectra of 54 common metabolites at varied pH (6.0 to 8.0 in 0.5 step increments) and temperature (290 K to 308 K) to quantify chemical shift variability to facilitate automated metabolite assignments. Our results illustrate the fundamental challenges with accurately assigning NMR peaks under varied environmental conditions prevalent in complex mixtures. Phosphorylated metabolites showed a larger variation in chemical shifts due to pH, whereas; amino acids showed a higher variation due to temperature. Mixtures of phosphorous compounds showed a consistently poor reliability in achieving an accurate assignment. Phosphorylated cholines, amino acids, and glycerols yielded a 40 % false negative rate for 7 out of 9 mixture conditions. Amino acids had a false negative rate of 57 % at 298 K and pH 8. Our results demonstrate that the automated assignments of complex biofluid mixtures require an expert to intervene to confirm the accuracy of metabolite assignments. Our analysis also indicates the need for reference databases to include spectra under a variety of conditions that includes mixtures and a range of pH and temperature to improve the accuracy and reproducibility of metabolite assignments.

Li P., Su M., Chatterjee M., Lämmerhofer M.

Monitoring the glycolysis pathway remains an analytical challenge as most metabolites involved are sugar phosphates. Structural similarity, instability, high polarity, and rich negative charges of sugar phosphates make LC-MS based analysis challenging. Here, we developed an improved workflow integrating uniformly 13C-labeled yeast metabolite extract, TiO2-based enrichment, differential stable isotope labeling phosphate methylation, porous graphic carbon column, and selected reaction monitoring acquisition. Uniformly 13C labeled yeast metabolite extract was used as internal standards while differential stable isotope labeled sugar phosphates worked as calibrants. The established method was validated in human plasma, platelet and cultured HeLa cells. The limits of quantification ranged between 0.25 and 0.54 pmol on column. The method was adapted and its applicability tested for human platelets in which activation with collagen-related peptide (CRP) clearly showed the upregulation of some SPx metabolites. The results document that this newly established method can be successfully used to monitor glycolysis in different biological samples. As an extension, more phosphorylated and carboxylated metabolites from the central carbon metabolism (pentose phosphate cycle, TCA cycle) were tested as well. This method showed superior performance, especially for multiple phosphorylated and carboxylated metabolites. For quantitative purpose, the concept of SPx in three sets (12C-analytes, U-13C-IS, deuterated calibrants) has the potential to be adapted for more anionic metabolites.

Palyzová A., Guschina I.A., Řezanka T.

Phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in bacteria. However, the chirality of the carbon atom on glycerol phosphate is different between the three kingdoms, Archaea, Bacteria, and Eukarya. Archaea membranes consist of phospholipids with glycerol-1-phosphate (G1P) in the S configuration, whereas phospholipids of the other two kingdoms contain glycerol-3-phosphate (G3P) having R stereochemistry. In the present study, GC/MS and LC/MS methods sensitively detected G3P and G1P from four bacterial strains (Bacillus amyloliquefaciens, B. subtilis, Clavibacter michiganensis, and Geobacillus stearothermophilus). Strain selection was carried out based on a GenBank search that revealed bacterial sequences associated with both enzymes involved in glycerol-phosphate synthesis, i.e., glycerol-3-phosphate dehydrogenase and glycerol-1-phosphate dehydrogenase. The detection of G1P and G3P was made by comparing the retention times of synthetic standards with those of analyzed samples. The structures of both glycerol phosphates were confirmed by selected ion monitoring (SIM) at m/z 171.006. The total concentration of G3P and G1P was around 30 µM, with a ratio of G3P to G1P of 4:1. We showed that PG was the most abundant phospholipid in all four bacteria by using the following analytical techniques and chromatographic modes: hydrophilic interaction liquid chromatography (HILIC), reversed-phase high-performance liquid chromatography high-resolution electrospray ionization tandem mass spectrometry (RP-HPLC/HR-ESI tandem MS) in negative and positive ionization modes, and an enzymatic cleavage by phospholipase C. By using chiral chromatography, the presence of both enantiomers in the glycerol backbone of some molecular species of PG was revealed. These results allow us to conclude that the bacteria examined here produce both enantiomer glycerol phosphates.

Essuman K., Milbrandt J., Dangl J.L., Nishimura M.T.

In the 20th century, researchers studying animal and plant signaling pathways discovered a protein domain that is shared across diverse innate immune systems: the Toll/interleukin-1/resistance gene (TIR) domain. The TIR domain is found in several protein architectures and was defined as an adaptor that mediates protein-protein interactions in animal innate immunity and developmental signaling pathways. However, studies of nerve degeneration in animals—and subsequent breakthroughs in plant, bacterial, and archaeal systems—revealed that TIR domains possess enzymatic activities. We provide a synthesis of TIR functions and the role of various related TIR enzymatic products in evolutionarily diverse immune systems. These studies may ultimately guide interventions that would span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases.

Wishart D.S., Cheng L.L., Copié V., Edison A.S., Eghbalnia H.R., Hoch J.C., Gouveia G.J., Pathmasiri W., Powers R., Schock T.B., Sumner L.W., Uchimiya M.

Metabolomics investigates global metabolic alterations associated with chemical, biological, physiological, or pathological processes. These metabolic changes are measured with various analytical platforms including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). While LC-MS methods are becoming increasingly popular in the field of metabolomics (accounting for more than 70% of published metabolomics studies to date), there are considerable benefits and advantages to NMR-based methods for metabolomic studies. In fact, according to PubMed, more than 926 papers on NMR-based metabolomics were published in 2021—the most ever published in a given year. This suggests that NMR-based metabolomics continues to grow and has plenty to offer to the scientific community. This perspective outlines the growing applications of NMR in metabolomics, highlights several recent advances in NMR technologies for metabolomics, and provides a roadmap for future advancements.

Wohlgemuth R., Littlechild J.

The biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules.

Mironov N., Atfi A., Razzaque M.S.

Aoki M., Vinokur J., Motoyama K., Ishikawa R., Collazo M., Cascio D., Sawaya M.R., Ito T., Bowie J.U., Hemmi H.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Fulmali A., Bharate S.S.

The salification and prodrug approaches modulate the physicochemical properties and absorption, distribution, metabolism, excretion, and toxicity parameters of drugs and lead candidates. The “phosphate” is one of the key counterions/promoiety used in the salt formation and prodrug synthesis. Salification with phosphoric acid enhances the aqueous solubility and thereby facilitates the administration of a drug by the parenteral route. Phosphate moiety in prodrug synthesis mainly improves permeability by lipophilic substitution. Histamine phosphate is the first phosphate salt, and hydrocortisone phosphate was the first prodrug approved by FDA in 1939 and 1952, respectively. The orange book enlists 12 phosphate salts and 17 phosphate prodrugs. Phosphate prodrugs, namely combretastatin A-4 diphosphate, combretastatin A-4 phosphate, lufotrelvir, TP-1287, pyridoxal phosphate, riboflavin phosphate, and psilocybin are clinical candidates. This review focuses on the FDA-approved phosphate salts and prodrugs from 1939 to 2021. The biopharmaceutical advantage of phosphate salts and prodrugs over the parent molecule is also deliberated.

Yu D., Song W., Tan E.Y., Liu L., Cao Y., Jirschitzka J., Li E., Logemann E., Xu C., Huang S., Jia A., Chang X., Han Z., Wu B., Schulze-Lefert P., et. al.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Scott K.A., Ropek N., Melillo B., Schreiber S.L., Cravatt B.F., Vinogradova E.V.

Chirality is an inherent aspect of biology, and interactions between biomolecules are often influenced by stereochemistry and topographic complexity. This has implications for how small-molecule libraries are assembled for screening campaigns in chemical biology and drug discovery. Here we review the state of the field in the context of harnessing chirality as a source of chemical information at the chemistry-biology interface. We further highlight the emergence of screening libraries containing stereoisomeric sets of compounds and the concept of using stereoselectivity of phenotype and/or target engagement as a way to prioritize actionable targets and streamline the identification of selective and potent modulators of disease-relevant biomolecules. The chemical information density of FDA-approved drugs and the effect of stereochemistry on molecular complexity are reported. Finally, axial chirality and atroposelectivity are discussed as potential expansions of the aforementioned concepts.

Zhang P., Gao M., Zhang Z., Xu F.