A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

Identification of d -arabinan-degrading enzymes in mycobacteria

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall

A Click Chemistry Approach to 5,5′-Disubstituted-3,3′-Bisisoxazoles from Dichloroglyoxime and Alkynes: Luminescent Organometallic Iridium and Rhenium Bisisoxazole Complexes

5,5′-Disubstituted-3,3′-bisisoxazoles are prepared in one step by the dropwise addition of aqueous potassium hydrogen carbonate to a mixture of dichloroglyoxime and terminal alkynes. The reaction exhibits a striking preference for the 5,5′-disubstituted 3,3′-bisisoxazole over the 4,5′-regioisomer. Organometallic iridium and rhenium bisisoxazole complexes are luminescent with emission wavelengths varying depending upon the identity of the 5,5′-substituent (phenyl, butyl)

A Click Chemistry Approach to 5,5′-Disubstituted-3,3′-Bisisoxazoles from Dichloroglyoxime and Alkynes: Luminescent Organometallic Iridium and Rhenium Bisisoxazole Complexes

5,5′-Disubstituted-3,3′-bisisoxazoles are prepared in one step by the dropwise addition of aqueous potassium hydrogen carbonate to a mixture of dichloroglyoxime and terminal alkynes. The reaction exhibits a striking preference for the 5,5′-disubstituted 3,3′-bisisoxazole over the 4,5′-regioisomer. Organometallic iridium and rhenium bisisoxazole complexes are luminescent with emission wavelengths varying depending upon the identity of the 5,5′-substituent (phenyl, butyl)

A Click Chemistry Approach to 5,5′-Disubstituted-3,3′-Bisisoxazoles from Dichloroglyoxime and Alkynes: Luminescent Organometallic Iridium and Rhenium Bisisoxazole Complexes

5,5′-Disubstituted-3,3′-bisisoxazoles are prepared in one step by the dropwise addition of aqueous potassium hydrogen carbonate to a mixture of dichloroglyoxime and terminal alkynes. The reaction exhibits a striking preference for the 5,5′-disubstituted 3,3′-bisisoxazole over the 4,5′-regioisomer. Organometallic iridium and rhenium bisisoxazole complexes are luminescent with emission wavelengths varying depending upon the identity of the 5,5′-substituent (phenyl, butyl)

Acetylation of Trehalose Mycolates Is Required for Efficient MmpL-Mediated Membrane Transport in Corynebacterineae

Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum<i> NCgl2759</i> resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The <i>in vitro</i> synthesis of AcTMCM was dependent on acetyl-CoA, whereas <i>in vivo</i> [¹⁴C]-acetate pulse–chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the Δ<i>NCgl2759</i> mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel <u>T</u>MCM <u>m</u>ycolyl <u>a</u>cetyl<u>t</u>ransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species