Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

ABSTRACT Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis , this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene ( ipdAB ) and an acyl-CoA reductase gene ( ipdC ). In the presence of cholesterol, Δ ipdC and Δ ipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα- H -4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and ( R )-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A Δ fadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by Δ ipdF and Δ echA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a Δ ipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. IMPORTANCE Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis , this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics.

Yields of hydrogen peroxide from the reaction of hydroxyl radical with organic compounds in solution and ice

Hydrogen peroxide (HOOH) is a significant oxidant in atmospheric condensed phases (e.g., cloud and fog drops, aqueous particles, and snow) that also photolyzes to form hydroxyl radical (•OH). •OH can react with organics in aqueous phases to form organic peroxyl radicals and ultimately reform HOOH, but the efficiency of this process in atmospheric aqueous phases, as well as snow and ice, is not well understood. We investigate HOOH formation from •OH attack on 10 environmentally relevant organic compounds: formaldehyde, formate, glycine, phenylalanine, benzoic acid, octanol, octanal, octanoic acid, octanedioic acid, and 2-butoxyethanol. Liquid and ice samples with and without nitrate (as an •OH source) were illuminated using simulated solar light, and HOOH formation rates were measured as a function of pH and temperature. For most compounds, the formation rate of HOOH without nitrate was the same as the background formation rate in blank water (i.e., illumination of the organic species does not produce HOOH directly), while formation rates with nitrate were greater than the water control (i.e., reaction of •OH with the organic species forms HOOH). Yields of HOOH, defined as the rate of HOOH production divided by the rate of •OH production, ranged from essentially zero (glycine) to 0.24 (octanal), with an average of 0.12 ± 0.05 (95 % CI). HOOH production rates and yields were higher at lower pH values. There was no temperature dependence of the HOOH yield for formaldehyde or octanedioic acid between −5 to 20 °C and ice samples had approximately the same HOOH yield as the aqueous solutions. In contrast, HOOH yields in formate solutions were higher at 5 and 10 °C compared to −5 and 20 °C. Yields of HOOH in ice for solutions containing nitrate and either phenylalanine, benzoate, octanal, or octanoic acid were indistinguishable from zero. Our HOOH yields were approximately half those found in previous studies conducted using γ-radiolysis, but this difference might be due to the much lower (and more environmentally relevant) •OH formation rates in our experiments.

Synthesis and Crystal Structure of a New Cd(II) Coordination Polymer Based on a 1,10-Phenanthroline Derivative

The new coordination polymer [Cd(oct)(L)] (H2oct = octanedioic acid and L = 2-(4-fluorophenyl)-1H-imidazo- [4,5-f][1,10]phenanthroline) has been synthesized under hydrothermal condition and characterized by elemental analysis, IR spectroscopy and single-crystal X-ray diffraction. Crystal data: C27H23CdFN4O4, monoclinic, space group C2/c, a = 21.391(4), b = 18.180(4), c = 14.031(3) Å , β = 116.50(3), V = 4883(2) Å3, Z = 8. The flexible oct dianions bridge the Cd(II) cations to yield a double-chain structure. The L ligands are attached on both sides of the chains, and their π-π interactions between neighboring chains result in two-dimensional supramolecular layers

Yields of hydrogen peroxide from the reaction of hydroxyl radical with organic compounds in solution and ice

Hydrogen peroxide (HOOH) is a significant oxidant in atmospheric condensed phases (e.g., cloud and fog drops, aqueous particles, and snow) that photolyzes to form hydroxyl radical (·OH). ·OH can react with organics in aqueous phases to form organic peroxyl radicals and ultimately reform HOOH, but the efficiency of this process in atmospheric aqueous phases, as well as snow and ice, is not well understood. We investigate HOOH formation from ·OH radical attack of 10 environmentally relevant organic compounds: formaldehyde, formate, glycine, phenylalanine, benzoic acid, octanol, octanal, octanoic acid, octanedioic acid, and 2-butoxyethanol. Liquid and ice samples with and without nitrate (as an ·OH source) were illuminated using simulated solar light, and HOOH formation rates were measured as a function of pH and temperature. For most compounds, the formation rate of HOOH without nitrate were the same as the background formation rate in blank water (i.e., illumination of the organic species does not produce HOOH directly), while formation rates with nitrate were greater than the water control (i.e., reactions of OH with the organic species forms HOOH). Yields of HOOH, defined as the rate of HOOH production divided by the rate of ·OH production, ranged from essentially zero (glycine) to 0.24 (octanal), with an average of 0.12 ± 0.05 (95% CI). HOOH production rates and yields were higher at lower pH values. There was no temperature dependence of the HOOH yield for formaldehyde or octanedioic acid between −5 to 20 °C and ice samples had approximately the same HOOH yield as the aqueous solutions. In contrast, HOOH yields in formate solutions were higher at 5 and 10 °C compared to −5 and 20 °C. Yields of HOOH in ice for solutions containing nitrate and either phenylalanine, benzoate, octanal, or octanoic acid were indistinguishable from zero. Our HOOH yields were approximately half that found in previous studies conducted using γ-radiolysis, but this difference might be due to the much lower (and more environmentally relevant) ·OH formation rates in our experiments.

Average structure of the composite crystal urea/octanedioic acid at room temperature within the superspace formalism

The average structure of the composite urea/octanedioic acid has been refined using the superspace formalism [superspace group H′3121(00γ)00\bar{1}]. Modulation effects seem to be almost negligible. The guest substructure appears to be largely disordered and has been modelled using rigid units occupying 12 equiprobable different orientations inside the urea tunnels. Guest molecules are slightly tilted with respect to the tunnel axis favouring a stronger guest–guest intratunnel interaction.