Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S-Methyltransferase.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt-/- ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt-/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates.

Structural analysis of linear hydroxyproline-bound O-glycans of Chlamydomonas reinhardtii--conservation of the inner core in Chlamydomonas and land plants.

Linear hydroxyproline-bound O-glycans of the unicellular green alga Chlamydomonas reinhardtii were isolated from outer cell wall glycoproteins and their structure elucidated by chemical and spectroscopic methods. They consist exclusively of arabinose and galactose, the latter in the furanose form, unusual for plants. The first two arabinoses linked to hydroxyproline have the same anomeric configuration and linkage to each other as do the arabinosides isolated from land plants, suggesting that these two steps of hydroxyproline-O-glycosylation have been conserved during evolution. In a concomitant analysis of Hyp-bound O-tri- and tetraarabinans isolated from Arabidopsis thaliana, we were able to unambiguously identify the natural trans-4-l-Hyp-bound O-glycans and the artificially produced d-allo forms and to identify diagnostic signals in NMR spectra of these compounds for the first time.

In vivo labeling with stable isotopes as a tool for the identification of unidentified peaks in the metabolome analysis of Corynebacterium glutamicum by GC/MS.

Of the hyphenated techniques used for metabolic profiling of cell and tissue extracts, GC/MS is in some ways advantageous as it allows the simultaneous fingerprinting of chemically very different metabolites, and the electron impact mass spectra recorded in many cases lead to unambiguous identification of the compounds. However, prior to chromatography, the hydrophilic substances of the cell extracts have to be converted to vaporizable derivatives, the mass spectra of which often are not known or not listed in the available spectral libraries, even if they are derived from simple biochemicals. Thus, numerous chromatographic peaks remain as yet unidentified. Attempts to identify these peaks afford the acquisition of more data on these compounds. The value of in vivo labeling of metabolites with (13)C and (15)N for this purpose is described.

Metabolism of 4-hydroxyandrostenedione and 4-hydroxytestosterone: Mass spectrometric identification of urinary metabolites.

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.

The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation of fertile spores.

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.

Analysis of the iridals in rhizome extracts of Iris variegata Linn.

Fractionation of the lipid extract from rhizomes of Iris variegata Linn. resulted in the isolation of one new and four known iridals. The latter were identified by comparison with authentic standards and NMR-spectroscopic analyses as 21-hydroxyiridal 1, 21-hydroxy-10-deoxyiridal 2, 16-hydroxyiridal 3 and 22-methyl-gamma-cycloiridal 4. The (R) configuration of C21 in compound 2 was determined by the exciton chirality method. The same stereochemistry has previously been found for the appropriate moieties in 1 and 3. Based on its spectroscopic properties the new triterpenoid was shown to be the 23-hydroxyiridal 5.

Iridal glycosides from Iris spuria (Zeal), cultivated in Egypt.

Fractionation of a methanol extract obtained from rhizomes of I. spuria (Zeal) resulted in the isolation of seven iridalglycosides 5a,b, 6a-c, 7, 8. The structures of the aglycons and the position of the glycosidic bonds were elucidated by spectroscopic analyses. The nature of the sugar moieties was determined as glucose from capillary GC and GC/MS analyses after hydrolysis of the compounds, reduction and derivatization of the resulting hexitols in comparison to authentic standards. Methylation analysis revealed the glycosyl-linkage composition. The number and position of the glucose residues was determined by ozonolysis.