Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.
The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ 1 H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional 1 H-13 C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by 1 H and 19 F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.
2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.2-Mercaptobenzothiazole (MBT) is the most important and most widely used member of the benzothiazole (BT) family. MBT is typically a rubber additive (16,17), but it has also other applications, such as inhibiting biocorrosion in cooling systems or in paper manufacturing (4). The annual MBT production in Western Europe is estimated to be excess of 40,000 tons.Direct discharges of MBT occur in effluents from factories producing and using MBT. Indirect sources of environmental contamination are mainly leachates from landfills where MBT is deposited and from rubber products (18). MBT can also be found in tannery wastewater (20). Finally, BTs have been found in urban runoff, in residential and highway road dust, and in urban particulate matter, most probably as a result of vehicle tire wear (18). The U.S. Environmental Protection Agency estimated that over 500 tons of MBT may be lost annually to the environment (22). Its toxicity towards microorganisms (3, 6, 7, 9), its allergenicity, resulting in serious dermatoses (11), and its potential mutagenic effects (12) make its presence in the environment of great concern.Information on the environmental fate of BT in the literature is scarce, and information on its biodegradative pathways is scarcer still. Several studies were first carried out in laboratories or in pilot-scale activated sludge systems in order to remove such a compound (5,20,21). These studies show that MBT is rather recalcitrant and is not completely mineralized when its concentration reaches a certain threshold. The metabolites observed under these conditions were benzothiazolylsulfonate and 2-methylthiobenzothiazole (MTBT) (10,20), the disulfide derivative of MBT (5).Only a few bacterial isolates have been shown to biotransform MBT. Drotar et al. (10) observed MBT-methylating activity in crude extracts of a variety of soil and water isolates, including a Corynebacterium sp., a Pseudomonas sp., and Escherichia coli, yielding the more stable methylated product MTBT, which accumulated in the medium.This paper reports studies of the biotransformation of MBT by Rhodococcus rhodochrous OBT18, isolated by De Wever et al. (8) from activated sludge from a wastewater treatment plant of an MBT-producing factory (Bayer, Antwerp, Belgium). This strain was previously shown to degrade BT, 2-hydroxybenzothiazole (OBT) (2, 8), and 2-aminobenzothiazole (ABT) (13). The main objective of this work was to identify the structure of the metabolites formed in order to establish the metabolic pathway of MBT for this strain. Because BT structures are difficult to analyze (due to the presence of N and S heteroatoms) and the metabolism of BT is almost unknown, various powerful...
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