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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 EgtE mechanistic model.
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f8: Proposed EgtE mechanistic model.

Mentions: EgtB-catalysis seems to be distinct from all currently known C-S bond formation chemistries. EgtB is a mononuclear non-heme iron enzyme and it was proposed that FeIV = O species are involved in this oxidative C-S bond formation process37387677. The proposed mechanistic models still await to be verified by future trapping and characterization of the proposed intermediates. Besides the novel C-S bond formation chemistry catalyzed by EgtB, the EgtE catalysis is equally intriguing. EgtE is a PLP-containing enzyme based on both bioinformatic sequence analysis and characterization of the purified protein. To explain all of the discoveries reported in this study, we proposed an EgtE mechanistic model (Fig. 8). Similar to other PLP-containing C-S lyases3435, the first step is the formation of the Schiff-base (17) between the PLP cofactor and the substrate (4), deprotonation of the Cys α-carbon leads to intermediate 18. The subsequent C-S bond cleavage produces a PLP-based adduct (19) and a sulfenic intermediate of hercynine (12), which is released from the active site into the solvent environment. Due to its instability, the disproportionation reaction between two molecules of 12 will lead to the formation of a thiol ester of thio-sulfinic acid 13, which is the compound detected in EgtE reaction when a reductant was not included in the reaction mixture (Fig. 4D). In the presence of DTT, ergothioneine sulfenic acid (12) will be reduced to ergothioneine (Fig. 4E). In the C-S lyase reaction of intermediate 18, besides sulfenic acid 13, a PLP-based intermediate 19 will also form. Similar to other C-S lyases35, the amine exchange between this intermediate and an EgtE active site lysine residue leads to the production of pyruvate (21) and ammonia as the side-products. This mechanistic model is consistent with the production of ergothioneine, pyruvate, and ammonia in a ratio of 1:1:1 based on the quantitative analysis of these three products. This model can also be used to explain the result when thio-ether 8 was used as substrate. When thio-ether 8 is used as substrate, the C-S lyase reaction from an intermediate analogous to intermediate 18 will produce ergothioneine directly. Thus, when thio-ether 8 is used as substrate, no reduction is required. In addition, the successful isolation of the adduct (16) between the proposed sulfenic acid intermediate 12 and dimedone provided further evidence supporting the proposed EgtE reaction mechanism. Our EgtE product profile is also consistent with the behaviors of ergothioneine under oxidative conditions, in which sulfenic acid intermediate was also suggested to be involved42. The successful trapping and characterization of sulfenic acid intermediate in EgtE catalysis provide evidences supporting the involvement of ergothioneine sulfenic acid as a key intermediate when ergothioneine is exposed to oxidative conditions as suggested by Servillo et al.42


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 EgtE mechanistic model.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Proposed EgtE mechanistic model.
Mentions: EgtB-catalysis seems to be distinct from all currently known C-S bond formation chemistries. EgtB is a mononuclear non-heme iron enzyme and it was proposed that FeIV = O species are involved in this oxidative C-S bond formation process37387677. The proposed mechanistic models still await to be verified by future trapping and characterization of the proposed intermediates. Besides the novel C-S bond formation chemistry catalyzed by EgtB, the EgtE catalysis is equally intriguing. EgtE is a PLP-containing enzyme based on both bioinformatic sequence analysis and characterization of the purified protein. To explain all of the discoveries reported in this study, we proposed an EgtE mechanistic model (Fig. 8). Similar to other PLP-containing C-S lyases3435, the first step is the formation of the Schiff-base (17) between the PLP cofactor and the substrate (4), deprotonation of the Cys α-carbon leads to intermediate 18. The subsequent C-S bond cleavage produces a PLP-based adduct (19) and a sulfenic intermediate of hercynine (12), which is released from the active site into the solvent environment. Due to its instability, the disproportionation reaction between two molecules of 12 will lead to the formation of a thiol ester of thio-sulfinic acid 13, which is the compound detected in EgtE reaction when a reductant was not included in the reaction mixture (Fig. 4D). In the presence of DTT, ergothioneine sulfenic acid (12) will be reduced to ergothioneine (Fig. 4E). In the C-S lyase reaction of intermediate 18, besides sulfenic acid 13, a PLP-based intermediate 19 will also form. Similar to other C-S lyases35, the amine exchange between this intermediate and an EgtE active site lysine residue leads to the production of pyruvate (21) and ammonia as the side-products. This mechanistic model is consistent with the production of ergothioneine, pyruvate, and ammonia in a ratio of 1:1:1 based on the quantitative analysis of these three products. This model can also be used to explain the result when thio-ether 8 was used as substrate. When thio-ether 8 is used as substrate, the C-S lyase reaction from an intermediate analogous to intermediate 18 will produce ergothioneine directly. Thus, when thio-ether 8 is used as substrate, no reduction is required. In addition, the successful isolation of the adduct (16) between the proposed sulfenic acid intermediate 12 and dimedone provided further evidence supporting the proposed EgtE reaction mechanism. Our EgtE product profile is also consistent with the behaviors of ergothioneine under oxidative conditions, in which sulfenic acid intermediate was also suggested to be involved42. The successful trapping and characterization of sulfenic acid intermediate in EgtE catalysis provide evidences supporting the involvement of ergothioneine sulfenic acid as a key intermediate when ergothioneine is exposed to oxidative conditions as suggested by Servillo et al.42

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