Discovery and Characterization of the Metallopterin-Dependent Ergothioneine Synthase from Caldithrix abyssi

Ergothioneine is a histidine derivative with a 2-mercaptoimidazole side chain and a trimethylated α-amino group. Although the physiological function of this natural product is not yet understood, the facts that many bacteria, some archaea, and most fungi produce ergothioneine and that plants and animals have specific mechanisms to absorb and distribute ergothioneine in specific tissues suggest a fundamental role in cellular life. The observation that ergothioneine biosynthesis has emerged multiple times in molecular evolution points to the same conclusion. Aerobic bacteria and fungi attach sulfur to the imidazole ring of trimethylhistidine; via; an O; 2; -dependent reaction that is catalyzed by a mononuclear non-heme iron enzyme. Green sulfur bacteria and archaea use a rhodanese-like sulfur transferase to attach sulfur; via; oxidative polar substitution. In this report, we describe a third unrelated class of enzymes that catalyze sulfur transfer in ergothioneine production. The metallopterin-dependent ergothioneine synthase from; Caldithrix abyssi; contains an N-terminal module that is related to the tungsten-dependent acetylene hydratase and a C-terminal domain that is a functional cysteine desulfurase. The two modules cooperate to transfer sulfur from cysteine onto trimethylhistidine. Inactivation of the C-terminal desulfurase blocks ergothioneine production but maintains the ability of the metallopterin to exchange sulfur between ergothioneine and trimethylhistidine. Homologous bifunctional enzymes are encoded exclusively in anaerobic bacterial and archaeal species

Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme

Molecular oxygen (O 2 ) is a sustainable oxidation reagent. O 2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O 2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research. [1] Activation of ground-state diradical O 2 can occur either via conversion to singlet oxygen or by one-electron reduction to superoxide. Many enzymes facilitate activation of O 2 by direct fomation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O 2 juxtaposed, but not coordinated to the catalytic Cu I . Isostructural complexes that contain Ag I instead of Cu I or nitric oxide instead of O 2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O 2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes

Disk Cooling and Wind Lines As Seen In the Spectral Line Evolution of V960 Mon

We follow up our photometric study of the post-outburst evolution of the FU Ori object V960 Mon with a complementary spectroscopic study at high dispersion that uses time series spectra from Keck/HIRES. Consistent with the photometric results reported in Carvalho et al. 2023, we find that the spectral evolution of V960 Mon corresponds to a decrease in the temperature of the inner disk, driven by a combination of decreasing accretion rate and increasing inner disk radius. We also find that although the majority of the absorption lines are well-matched by our accretion disk model spectrum, there are several strong absorption line families and a few emission lines that are not captured by the model. By subtracting the accretion disk model from the data at each epoch, we isolate the wind/outflow components of the system. The residuals show both broad and highly blueshifted profiles, as well as narrow and only slightly blueshifted profiles, with some lines displaying both types of features.Comment: 21 pages, 19 figures, accepted by Ap

In Vitro Production of Ergothioneine Isotopologues

Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues

Repetitive proteins from the flagellar cytoskeleton of African trypanosomes are diagnostically useful antigens

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection

Repetitive proteins from the flagellar cytoskeleton of African trypanosomes are diagnostically useful antigens

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detectio

Spin-dependent thermoelectric transport in HgTe/CdTe quantum wells

We analyze thermally induced spin and charge transport in HgTe/CdTe quantum wells on the basis of the numerical non-equilibrium Green's function technique in the linear response regime. In the topologically non-trivial regime, we find a clear signature of the gap of the edge states due to their finite overlap from opposite sample boundaries -- both in the charge Seebeck and spin Nernst signal. We are able to fully understand the physical origin of the thermoelectric transport signatures of edge and bulk states based on simple analytical models. Interestingly, we derive that the spin Nernst signal is related to the spin Hall conductance by a Mott-like relation which is exact to all orders in the temperature difference between the warm and the cold reservoir.Comment: 11 pages, 13 figures, submitted to PR

Structure of formylglycine-generating enzyme in complex with copper and a substrate reveals an acidic pocket for binding and activation of molecular oxygen

The formylglycine generating enzyme (FGE) catalyzes oxidative conversion of specific peptidyl-cysteine residues to formylglycine. FGE mediates O; 2; -activation and hydrogen-atom abstraction in an active site that contains Cu(i) coordinated to two cysteine residues. Similar coordination geometries are common among copper-sensing transcription factors and copper-chaperone but are unprecedented among copper-dependent oxidases. To examine the mechanism of this unusual catalyst we determined the 1.04 Å structure of FGE from; Thermomonospora curvata; in complex with copper and a cysteine-containing peptide substrate. This structure unveils a network of four crystallographic waters and two active site residues that form a highly acidic O; 2; -binding pocket juxtaposed to the trigonal planar tris-cysteine coordinated Cu(i) center. Comparison with structures of FGE in complex with Ag(i) and Cd(ii) combined with evidence from NMR spectroscopy and kinetic observations highlight several structural changes that are induced by substrate binding and prime the enzyme for O; 2; -binding and subsequent activation

Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattl

Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle