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Low-molecular-weight (LMW) thiols play critical roles in maintaining redox buffer systems required for normal biological function. Glutathione (GSH) represents the most common LMW thiol found in Nature, but Gram-positive bacteria utilize bacillithiol (BSH) or mycothiol (MSH). Nitroxyl (HNO) can influence bacterial transcription through persulfide formation, a biological phenomenon that prompts the examination of the reactions of HNO with these LMW thiols. The development and application of colorimetric and enzymatic (Bacillus subtilis thioredoxin assay) methods combined with mass spectrometry of reaction products show the unique reactivity of BSH to favor sulfinamide adduct formation upon equimolar reaction with HNO. The reaction profile with GSH results in nearly equal distribution between sulfinamide:disulfide, whereas reaction with MSH only yields disulfide. These varied results led to the preparation of a group of BSH and MSH analogs, and their reactions with HNO reveal the requirement for a free amine group for sulfinamide formation. The thiol and amine group pKa's appear critical for sulfinamide generation, with the thiolate acting as a nucleophile to attack HNO and the ammonium donating a proton to facilitate water loss from the N-hydroxysulfenamide intermediate. Furthermore, the B. subtilis thioredoxin system efficiently reduces BSSB with a calculated KM_BSSB = 34 ± 3 μM and Vmax = 152 ± 3.4 nmol/min/nmol TrxR (kcat = 2.5 s−1), but does not reduce bacillithiol sulfinamide. Similarly, this thioredoxin reduces MSSM with a calculated KM_MSSM of 9 ± 2.1 μM and Vmax of 103 ± 7.1 nmol/min/nmol TrxR (kcat = 1.7 s−1). Bacillithiol possesses a unique structure that allows a rapid reaction with HNO to form a stable product that may provide a basis for antibiotic development or clues for further biological roles of nitroxyl.
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Sulfinamide Formation from the Reaction of Bacillithiol and Nitroxyl
Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/ enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli’s salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine−cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N-hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol.
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- Award ID(s):
- 1716535
- PAR ID:
- 10567884
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Chemical Biology
- Volume:
- 18
- Issue:
- 12
- ISSN:
- 1554-8929
- Page Range / eLocation ID:
- 2524 to 2534
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acschembio.3c00526
