Chelating agent

Propanethiol (PT) is a hazardous pollutant that poses risks to both the environment and human well-being. Pseudomonas putida S-1 has been identified as a microorganism capable of utilizing PT as its sole carbon source. However, the metabolic pathway responsible for PT degradation in P. putida S-1 has remained poorly understood, impeding its optimization and practical application. In this study, we investigated the catabolic network involved in PT desulfurization with P. putida S-1 and identified key gene modules crucial to this process. Notably, propanethiol oxidoreductase (PTO) catalyzes the initial degradation of PT, a pivotal step for P. putida S-1's survival on PT. PTO facilitates the oxidation of PT, resulting H2S, H2O2, and propionaldehyde (PA). Catalase-peroxidase catalyzes the conversion of H2O2 to oxygen and water, while PA undergoes gradual conversion to Succinyl-CoA, which is subsequently utilized in the tricarboxylic acid cycle. H2S is digested in a comprehensive desulfurization network where sulfide-quinone oxidoreductase (SQOR) predominantly converts it to sulfane sulfur. The transcriptome analysis suggests that sulfur can be finally converted to sulfite or sulfate and exported out of the cell. [1]

The Propanethiol (PT) degradation capacity of P. putida S-1 was enhanced by increasing the transcription level of PTO and SQOR genes in vivo.IMPORTANCEThis work investigated the PT catabolism pathway in Pseudomonas putida S-1, a microorganism capable of utilizing PT as the sole carbon source. Critical genes that control the initiation of PT degradation were identified and characterized, such as pto and sqor. By increasing the transcription level of pto and sqor genes in vivo, we have successfully enhanced the PT degradation efficiency and growth rate of P. putida S-1. This work does not only reveal a unique PT degradation pathway but also highlights the potential of enhancing the microbial desulfurization process in the bioremediation of thiol-contaminated environment. [1]

Strains and plasmids [1]
Strain Pseudomonas putida S-1 was originally isolated from active sludge and grew at 30°C and 180 rpm for  Propanethiol (PT) degradation. Strain E. coli DH5α was used as host for plasmid construction. Strain E. coli BL21 (DE3) was used as host for protein expression. The auxotroph strain E. coli WM3064 was used for genome editing, and it was cultivated in LB with a supplementation of 2,6-diaminopimelic acid (57 mg/L).

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Enzyme activity assays [1]
Steady-state kinetics of PTO and SQOR was analyzed according to the classic Michaelis-Menten equation. The protein concentration of PTO and SQOR was diluted to 100 µM for catalytic reaction. The concentration of FAD and CoQ1 was also set to 100 µM. The catalytic reactions were conducted at certain substrate concentrations, and the initial rate was calculated for generation of MM plot. The data collected was fitted by using the software Graph Pad Prism version 8.

[1]. The critical roles of propanethiol oxidoreductase and sulfide-quinone oxidoreductase in the propanethiol catabolism pathway in Pseudomonas putida S-1. Appl Environ Microbiol . 2024 Feb 21;90(2):e0195923.

The degradation of volatile organic sulfur compounds (VOSCs) typically exhibits high substrate specificity. Previous studies on the biodegradation of thiols have primarily focused on the degradation of methanethiol; however, the complete catabolistic pathway has not been characterized in detail. This study delves into the finely regulated catabolistic mechanism network of propanethiol in Pseudomonas putida S-1 and identifies key gene modules initiating propanethiol desulfurization. Based on experimental observations, propanethiol catabolism in Pseudomonas putida S-1 is initiated by propanethiol oxidase (PTO, S1GL003403), with the reaction products H₂O₂, H₂S, and propanal subsequently catalyzed by thiosulfate esterases (SQOR, S1GL000007 and S1GL003435) and their downstream genes. In the BRENDA enzyme database (https://www.brenda-enzymes.org/index.php), thiol-based oxidases are classified into two classes: thiol oxidases (EC 1.8.3.2) and methanethiol oxidases (MTOs) (EC 1.8.3.4). Thiol oxidases convert thiols to their corresponding thioethers, while MTOs are limited to only one substrate—methanethiol. For example, the first reported MTO enzyme was isolated from the bacterium Hyphomicrobium sp. VS, with a Km value of approximately 0.3 µM. Human MTOs catalyze the formation of sulfides from methanethiol using a similar mechanism, with an apparent Km value of 1.8 nM; such a low Km value may help prevent the toxicity of methanethiol in humans. Although these MTO enzymes can catalyze the formation of sulfides from methanethiol with relatively high efficiency, their substrate range is narrow. For example, propanethiol cannot be catalyzed by them. PTOs exhibit high substrate specificity and selectivity. Observations revealed that Pseudomonas putida S-1, from which PTO was isolated, could not grow using methanethiol (MT) as the carbon source alone. If PTO can degrade MT, it would be expected that the bacteria would use the degradation products as a nutrient source. The limited substrate range of MTO or PTO suggests that thiol-based oxidases are likely to exhibit significant substrate specificity. The discovery of PTO fills an important gap in the existing enzyme library, suggesting its potential application value in the bioremediation of PT pollution. [1]
After the initial desulfurization step, the SQOR genes S1GL000007 and S1GL003435 catalyze the conversion of H₂S to thioalkyl sulfur, GSSH, or thiosulfate (Figure S8). Whole-genome sequencing also annotated three genes that may encode cysteine synthases, which can catalyze the production of cysteine from sulfides, but they were all downregulated under PT induction (Table S5). Given reports that cysteine oxidation generates reactive oxygen species (ROS), which can damage DNA and lead to mutations or cell death, its generation may not be a key step in the sulfur cycle in Pseudomonas putida S-1, despite its potential role as a bacterial carbon source. The SQOR product GSSH can be oxidized to sulfite by PDO, which can then be spontaneously oxidized or oxidized to sulfate by SDH. In Pseudomonas putida S-1, PT induction upregulates the expression of some related genes (e.g., S1GL000006) (Table S5). Sulfate can be expelled from Pseudomonas putida S-1 via sulfite efflux protein (TAUE) or reduced to sulfides by sulfite reductase (SRD). Due to the limited solubility of PT in water, it may form disulfides in the environment and enter the cell. Unlike tauE, the srd gene is significantly downregulated in Pseudomonas putida S-1 (Table S5). Sulfite can be converted to thiosulfate by thiosulfate-transferase, and thiosulfate can be further converted to tetrathiosulfate. However, the expression of the SfnB family sulfur-acquisition oxidoreductase (SFNB), which catalyzes this reaction, is significantly downregulated, indicating that tetrathiosulfate is not a favorable metabolite for PT catabolism. Propionaldehyde produced by PTO can be converted to propionic acid by aldehyde dehydrogenase. Propionic acid is converted to propionyl-CoA (CoA) by propionyl-CoA transferase. Finally, propionyl-CoA is converted to succinyl-CoA by propionyl-CoA:succinyl-CoA transferase and enters the tricarboxylic acid cycle (Figure S8). [1] In summary, we successfully discovered that the key gene modules S1GL003403 (pto) and S1GL003435 (sqor) are responsible for the initial catabolism of the PT catabolism pathway in Pseudomonas putida S-1. When the transcriptional levels of the pto and sqor genes are increased, the degradation capacity of PT by Pseudomonas putida S-1 is significantly enhanced. These findings provide new insights into enhancing microbial desulfurization processes and propose more strategies. [1] This study investigated the PT catabolic pathway in Pseudomonas putida S-1, which is able to utilize PT as its sole carbon source. We identified and characterized key genes controlling the initiation of PT degradation, such as pto and sqor. By increasing the transcriptional levels of the pto and sqor genes in vivo, we successfully enhanced the PT degradation efficiency and growth rate of Pseudomonas putida S-1. This work not only reveals a unique PT degradation pathway but also highlights the potential of enhancing microbial desulfurization processes in the bioremediation of mercaptan-contaminated environments.

463.16

C, 54.30; H, 7.59; Cl, 15.26; N, 9.05; S, 13.80

675825-78-2

675825-78-2 (HCl); 189950-11-6

16752681

Typically exists as solid at room temperature

4

5

10

28

434

4

HZDNFBVWGKDFIP-GCMGGMMESA-N

InChI=1S/C21H34ClN3S2.ClH/c1-24-18-6-7-21(24)20(15-25(11-13-27)10-8-23-9-12-26)19(14-18)16-2-4-17(22)5-3-16;/h2-5,18-21,23,26-27H,6-15H2,1H3;1H/t18-,19+,20-,21+;/m0./s1 Create Date: 2007-10-29

2-[2-[[(1R,2R,3S,5S)-3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]octan-2-yl]methyl-(2-sulfanylethyl)amino]ethylamino]ethanethiol;hydrochloride

Trodat 1; DLK3EG08UH; UNII-DLK3EG08UH; 675825-78-2; Ethanethiol, 2-((2-((((1R,2R,3S,5S)-3-(4-chlorophenyl)-8-methyl-8-azabicyclo(3.2.1)oct-2-yl)methyl)(2-mercaptoethyl)amino)ethyl)amino)-, hydrochloride (1:1)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)