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Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

Song H, Hu W, Naowarojna N, Her AS, Wang S, Desai R, Qin L, Chen X, Liu P - Sci Rep (2015)

Bottom Line: In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain.This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products.Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry, Boston University, Boston, MA 02215, USA.

ABSTRACT
Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

No MeSH data available.


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HMBC-NMR characterization of the sulfenic acid-dimedone adduct from the EgtE reaction.
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f6: HMBC-NMR characterization of the sulfenic acid-dimedone adduct from the EgtE reaction.

Mentions: To provide more evidence on the formation of an adduct between the proposed sulfenic acid intermediate 12 and 1,3-cyclohexanedione, the adduct was isolated and characterized. It has been reported that this type of adducts, the thio-ether derivative, was not stable under the acidic conditions50. Several attempted purifications using C18 HPLC methods failed due to the slight acidic environment during either the isolation or workup process. Later on, we discovered that the adduct can be isolated using cellulose resin under mild purification conditions. The adduct (16, Fig. 5) was fully characterized by 1H-NMR, 13C-NMR, 2-D NMR, and high-resolution mass spectrometry (Fig. 6 and Supplementary Fig. 17-21). 1H-13C correlations between H-2 and C-1, 3, 7 and 1H-13C correlations between H-5 and C-4, 6 in HMBC characterization supports compound 16 structural assignment (Fig. 6). Additional correlation between H-10 and C-8, 9 further supports the 1,3-cyclohexanedione portion in the compound 16. Moreover, the ratios for the integration of each peaks from 1H-NMR spectrum (Supplementary Fig. 17) and the high-resolution mass spectrometry data (m/z [M + H]+ found 340.1342, calcd. 340.1331, Supplementary Fig. 19) were also consistent with the assignment of compound 16 structure.


Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

Song H, Hu W, Naowarojna N, Her AS, Wang S, Desai R, Qin L, Chen X, Liu P - Sci Rep (2015)

HMBC-NMR characterization of the sulfenic acid-dimedone adduct from the EgtE reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493562&req=5

f6: HMBC-NMR characterization of the sulfenic acid-dimedone adduct from the EgtE reaction.
Mentions: To provide more evidence on the formation of an adduct between the proposed sulfenic acid intermediate 12 and 1,3-cyclohexanedione, the adduct was isolated and characterized. It has been reported that this type of adducts, the thio-ether derivative, was not stable under the acidic conditions50. Several attempted purifications using C18 HPLC methods failed due to the slight acidic environment during either the isolation or workup process. Later on, we discovered that the adduct can be isolated using cellulose resin under mild purification conditions. The adduct (16, Fig. 5) was fully characterized by 1H-NMR, 13C-NMR, 2-D NMR, and high-resolution mass spectrometry (Fig. 6 and Supplementary Fig. 17-21). 1H-13C correlations between H-2 and C-1, 3, 7 and 1H-13C correlations between H-5 and C-4, 6 in HMBC characterization supports compound 16 structural assignment (Fig. 6). Additional correlation between H-10 and C-8, 9 further supports the 1,3-cyclohexanedione portion in the compound 16. Moreover, the ratios for the integration of each peaks from 1H-NMR spectrum (Supplementary Fig. 17) and the high-resolution mass spectrometry data (m/z [M + H]+ found 340.1342, calcd. 340.1331, Supplementary Fig. 19) were also consistent with the assignment of compound 16 structure.

Bottom Line: In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain.This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products.Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry, Boston University, Boston, MA 02215, USA.

ABSTRACT
Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

No MeSH data available.


Related in: MedlinePlus