Insights into the Light Response of Skeletonema marinoi: Involvement of Ovothiol.

Diatoms are one of the most widespread groups of microalgae on Earth. They possess extraordinary metabolic capabilities, including a great ability to adapt to different light conditions. Recently, we have discovered that the diatom Skeletonema marinoi produces the natural antioxidant ovothiol B, until then identified only in clams. In this study, we investigated the light-dependent modulation of ovothiol biosynthesis in S. marinoi. Diatoms were exposed to different light conditions, ranging from prolonged darkness to low or high light, also differing in the velocity of intensity increase (sinusoidal versus square-wave distribution). The expression of the gene encoding the key ovothiol biosynthetic enzyme, ovoA, was upregulated by high sinusoidal light mimicking natural conditions. Under this situation higher levels of reactive oxygen species and nitric oxide as well as ovothiol and glutathione increase were detected. No ovoA modulation was observed under prolonged darkness nor low sinusoidal light. Unnatural conditions such as continuous square-wave light induced a very high oxidative stress leading to a drop in cell growth, without enhancing ovoA gene expression. Only one of the inducible forms of nitric oxide synthase, nos2, was upregulated by light with consequent production of NO under sinusoidal light and darkness conditions. Our data suggest that ovothiol biosynthesis is triggered by a combined light stress caused by natural distribution and increased photon flux density, with no influence from the daily light dose. These results open new perspectives for the biotechnological production of ovothiols, which are receiving a great interest for their biological activities in human model systems.

Thiols of intracellular pathogens. Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis.

Leishmania donovani, the causative agent of visceral leishmaniases, is an intracellular pathogen which proliferates within the host macrophages. Analysis of the thiol composition of L. donovani by means of the thiol-specific reagent, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, indicated that this organism produces substantial amounts of ovothiol A. This observation was further substantiated by HPLC of extracts of L. donovani after derivatization with bromobimane. L. donovani extracts contained a thiol, the bimane derivative of which had identical retention time and fluorescence quenching to a thiol from Crithidia fasciculata, which had previously been identified as ovothiol A. By comparison, the intracellular bacterial pathogen, Mycobacterium bovis, contained only one major low-molecular-mass thiol, which was assigned the trivial name mycothiol. The structure of the bimane derivative of mycothiol was solved by a combination of one- and two-dimensional 1H and 13C NMR spectroscopy. Spatial relationships in the molecule were further refined by NOE experiments and allowed identification of mycothiol as 1-D-myo-inositol-2-(N-acetyl-L-cysteinyl)amino-2-deoxy-alpha-D-glucopyra noside. This assignment was confirmed by positive-ion fast-atom-bombardment mass spectrometry which gave m/z = 677.6 Da and a sodiated species at 699.6 Da. Analysis of the dansylated hydrolysis products of performic-acid-oxidized mycothiol indicated the presence of 0.85 mol glucosamine and 1.02 mol cysteic acid/mol sulfhydryl groups. Crude extracts of M. bovis contained an enzyme which catalysed the NAD(P)H2-dependent reduction of mycothiol disulfide to the free thiol. Analysis of perchloric acid extracts of Mycobacterium tuberculosis H37RV indicated the presence of a thiol which comigrated with mycothiol, both as the free thiol and as the 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and bimane derivatives, on reverse-phase HPLC. The significance of these findings in terms of the evasion of the host defense mechanisms by leishmania parasites and mycobacteria is considered.

Identification of a major low-molecular-mass thiol of the trypanosomatid Crithidia fasciculata as ovothiol A. Facile isolation and structural analysis of the bimane derivative.

An unidentified low-molecular-mass thiol, U23, previously detected as the 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin derivative in extracts of the trypanosome Crithidia fasciculata, was purified as the bimane derivative. Resonances attributable to U23 were discerned from those of the bimane label by comparison of the 1H- and 13C-NMR spectra of monobromobimane and U23-bimane. The complete 1H- and 13C-NMR spectra of U23-bimane were assigned by means of 1H-1H correlation spectroscopy, 1H-13C correlation spectroscopy and 13C multiplicity determinations. The results indicated identity of U23 with 1-N-methyl-4-mercaptohistidine (ovothiol A), previously isolated from marine sources. This assignment was confirmed by NOE difference experiments, fast-atom-bombardment mass spectrometry of U23-bimane and ultraviolet/visible spectrophotometry of U23, which was isolated as the disulfide. The isolation of ovothiol A from a parasitic protozoan suggest that the 4-mercaptohistidines may have a wider distribution and function as antioxidant thiols than was hitherto realized.