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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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Proposed model to explain EgtE reaction outcomes when sulfoxide 4 was the substrate.
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f5: Proposed model to explain EgtE reaction outcomes when sulfoxide 4 was the substrate.

Mentions: After the EgtE substrate candidates (4 & 8) were synthesized, 1H-NMR was used to analyze EgtE reaction directly by monitoring the chemical shift of the hydrogen atoms on the hercynine imidazole side-chains in the substrate (4 or 8) and in the product, ergothioneine (5, Fig. 4). In 1H-NMR spectrum, the imidazole hydrogen in sulfoxide 4 has a chemical shift of 7.15 ppm, while the imidazole hydrogen in thio-ether 8 has a chemical shift of 6.89 ppm. Once the C-S bond is cleaved by EgtE, the resulting ergothioneine thiol-imidazole has a chemical shift of 6.67 ppm. Surprisingly, different from the assignment of EgtE function (Fig. 1)20, our 1H-NMR assay clearly indicates that EgtE recognizes both sulfoxide 4 and thio-ether 8 as substrates. This is consistent with the studies using M. smegmatis cell lysate reported by Khonde et al.36 However, many fine details were revealed when pure EgtE was used. When thio-ether 8 was used as the substrate, EgtE converts it to ergothioneine very efficiently (Fig. 4C). When sulfoxide 4 was used as substrate in KPi buffer, EgtE indeed accepts compound 4 as a substrate. However, 1H-NMR assay does not support the production of ergothioneine as the product (Fig. 4D). Instead, there are two signals at the imidazole ring hydrogen chemical shift region. Closer examination of the 1H-NMR spectrum of EgtE reaction mixture (Supplementary Fig. 5) revealed that there are also two sets of α-protons and imidazole ring protons. 2D-gCOSY NMR spectroscopy was then adopted to further characterize these two products. Both H-5 and H-5′ show correlation to the β-proton (H-3 and H-3′), which suggests two different kinds of histidine derivatives. Moreover, both sets of the α-protons (H-2 and H-2′) are correlated with the β-protons (H-3 and H-3′), which suggests that the histidine skeleton remains intact. When the products were isolated by HPLC, two products were identified, ergothioneine (5) and ergothioneine-2-sulfinic acid (14, Fig. 5). Both compounds were fully characterized by 1H-NMR, 13C-NMR, 2-D NMR, and high-resolution mass spectrometry (Supplementary Fig. 6-15).


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)

Proposed model to explain EgtE reaction outcomes when sulfoxide 4 was the substrate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Proposed model to explain EgtE reaction outcomes when sulfoxide 4 was the substrate.
Mentions: After the EgtE substrate candidates (4 & 8) were synthesized, 1H-NMR was used to analyze EgtE reaction directly by monitoring the chemical shift of the hydrogen atoms on the hercynine imidazole side-chains in the substrate (4 or 8) and in the product, ergothioneine (5, Fig. 4). In 1H-NMR spectrum, the imidazole hydrogen in sulfoxide 4 has a chemical shift of 7.15 ppm, while the imidazole hydrogen in thio-ether 8 has a chemical shift of 6.89 ppm. Once the C-S bond is cleaved by EgtE, the resulting ergothioneine thiol-imidazole has a chemical shift of 6.67 ppm. Surprisingly, different from the assignment of EgtE function (Fig. 1)20, our 1H-NMR assay clearly indicates that EgtE recognizes both sulfoxide 4 and thio-ether 8 as substrates. This is consistent with the studies using M. smegmatis cell lysate reported by Khonde et al.36 However, many fine details were revealed when pure EgtE was used. When thio-ether 8 was used as the substrate, EgtE converts it to ergothioneine very efficiently (Fig. 4C). When sulfoxide 4 was used as substrate in KPi buffer, EgtE indeed accepts compound 4 as a substrate. However, 1H-NMR assay does not support the production of ergothioneine as the product (Fig. 4D). Instead, there are two signals at the imidazole ring hydrogen chemical shift region. Closer examination of the 1H-NMR spectrum of EgtE reaction mixture (Supplementary Fig. 5) revealed that there are also two sets of α-protons and imidazole ring protons. 2D-gCOSY NMR spectroscopy was then adopted to further characterize these two products. Both H-5 and H-5′ show correlation to the β-proton (H-3 and H-3′), which suggests two different kinds of histidine derivatives. Moreover, both sets of the α-protons (H-2 and H-2′) are correlated with the β-protons (H-3 and H-3′), which suggests that the histidine skeleton remains intact. When the products were isolated by HPLC, two products were identified, ergothioneine (5) and ergothioneine-2-sulfinic acid (14, Fig. 5). Both compounds were fully characterized by 1H-NMR, 13C-NMR, 2-D NMR, and high-resolution mass spectrometry (Supplementary Fig. 6-15).

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