FAMIN Is a Multifunctional Purine Enzyme Enabling the Purine Nucleotide Cycle.

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods.

Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4-7 and a molecular mass range of 10-100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.

The validity of extrinsic stable isotopic labeling for mineral absorption studies in rats.

The use of extrinsic stable and radioisotopic labels (Fe, Zn, Cu and Se) was compared with the use of intrinsic labels by measuring label retention in rats. Saccharomyces cerevisiae (Hansen strain CBS 1171) was prepared intrinsically enriched with a stable isotope of iron, zinc, copper or selenium, and unenriched freeze-dried yeast was extrinsically labeled with the appropriate stable and/or radioisotope. Male Wistar rats, weighing 80-100 g and fed a purified diet, were given a test meal of one of the above labeled yeasts. Isotopic retention was determined by fecal monitoring. Retention of the stable isotopes was determined by thermal ionization quadruple mass spectrometry (TIQMS) and retention of the radioisotopes by counting feces in a whole-body counter. The results indicated that the behavior of the labels differed among the minerals, with copper as the only one in which the intrinsic and extrinsic stable isotopes were comparably retained. With zinc, retention of the extrinsic radiolabel and intrinsic label was similar, but retention of the extrinsic stable isotope label was higher. With iron, the intrinsic label had a significantly lower retention than the two extrinsic labels; with selenium, retention of all three labels was different, but these differences were not of a sufficient magnitude to conclude that extrinsic stable isotopic labelling is not valid. These results demonstrate that an extrinsic stable isotope label can be used for copper, selenium and inorganic iron, but that such a label is not valid for studies on zinc.

Detection of externally induced impairments in single bacterial cells by laser microbe mass analysis.

Applying the laser microbe mass analyzer method (LAMMA), mass spectra (fingerprints) were taken from single bacterial cells not treated or treated with high temperature, X-irradiation, isonicotinic acid hydrazide (INH), or diaminodiphenylsulfone (DDS). Spectra of treated cells ("M. lufu," M. tuberculosis H37Ra, E. coli) differ from those of controls in that the K+/Na+ ratio was smaller and in that the intensities of peaks with m/e greater than 100 were lower. From the results with M. leprae the possible application of this new method for monitoring the effectiveness of leprosy therapy is proposed.

[Studies on the lipids of "Mycobacterium gordonae" in comparison with those of "M. leprae" and of some other scotochromogenic mycobacteria (author's transl)]. / Etude des lipides de Mycobacterium gordonae comparativement à ceux de M. leprae et de quelques mycobactéries scotochromogènes.

The mycolic acids were isolated from Mycobacterium gordonae (strain ATCC 14470), and purified by thin layer chromatography. Three species were studied by mass spectrometry. The analogy between M. gordonae and M. leprae, based on the lack of tuberculostearic acid, was supported by the comparison of the structures of their mycolic acids. Succinct analyses of the lipids of three other scotochromogenic strains of mycobacteria, using thin layer chromatography and gas-liquid chromatography, were performed. These strains were more remote from M. gordonae than is M. leprae, as far as the lipid content is concerned.