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

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism

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

The Synthesis of 1,4-anhydro-α-D-Mannopyranose

Manuscript describing efforts, ultimately successful, to synthesize 1,4-anhydromannose. Full experimental details and spectroscopic characterization

Characterization of β-1,2-mannosyltransferase activity and inhibition. A) Extension of octyl α-d-mannoside by <i>L. mexicana Δgmp</i> β-1,2-mannosyltransferases.

<p>B) Fluorogram of HPTLC of 0.05 mM octyl α-d-mannopyranoside incubated with GDP-[³H]mannose in a membrane preparation of <i>L. mexicana Δgmp</i> with GDP-[³H]mannose; C) Michaelis-Menten plot (inset: Lineweaver-Burke replot) of rate versus [GDP-mannose] at saturating (50 mM) octyl α-d-mannopyranoside; D) Michaelis-Menten plot (inset: Lineweaver-Burke replot) of rate versus [octyl α-d-mannopyranoside] at saturating (10 mM) GDP-mannose; E) Fluorogram of HPTLC showing inhibition of β-1,2-mannosyltransferase-catalyzed extension of octyl α-d-mannopyranoside by GMP and GDP. M2–5 denote the oligomer length of oligomannosides formed from the substrate.</p

Synthesis of GMP-alkyne.

<p>a) (ⁱPr₂N)₂POCH₂CH₂CN, diisopropylammonium tetrazolide, CH₂Cl₂, 0°C→rt, 75 min, 46%; b) <i>i</i> 1<i>H</i>-tetrazole, propargyl alcohol, CH₂Cl₂, 0°C→rt, 15 min; <i>ii</i> 0.1 M I₂ in H₂O/pyridine/THF (2∶20∶80), 10 min, 57%; c) 1.0 M TBAF/THF, 45 min, Dowex 50W-X4 (Na⁺ form), 37%; d) Cu⁰, CuSO₄, DMSO/H₂O (9∶1), 50%.</p

Structures of biphenyl-based mono/diphosphonucleotide triazole adducts.

<p>Structures of biphenyl-based mono/diphosphonucleotide triazole adducts.</p

Identification and characterization of T47.

<p>A) Second-round screening of cherry-picked members of combinatorial library as inhibitors of extension of octyl α-d-mannopyranoside by <i>L. mexicana Δgmp</i> cell membranes containing β-1,2-mannosyltransferase. Assay conditions: 40 µM octyl α-D-mannopyranoside, 50 µM GDP-[³H]mannose, inhibitors at 1.27 mM, 15 min, 27°C. TLCs were developed in “solvent A”. “-ve” indicates reaction mixture that does not contain any inhibitor. M2,3 denote the oligomer length of oligomannosides formed from the substrate. B) Comparative IC₅₀ values of GMP and T47. Open squares denote GMP, closed triangles denote <b>T47</b>.</p

First-round screening of combinatorial library as inhibitors of extension of octyl α-D-mannopyranoside by <i>L. mexicana Δgmp</i> cell membranes containing β-1,2-mannosyltransferase.

<p>Assay conditions: 40 µM octyl α-d-mannopyranoside, 50 µM GDP-[³H]mannose, inhibitors at 4 mM, 15 min, 27°C. TLCs were developed in “solvent A”. “-ve” indicates reaction mixture that does not contain any inhibitor. M2–5 denote the oligomer length of oligomannosides formed from the substrate.</p

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)