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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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EgtE reactions under different in vitro conditions.
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f7: EgtE reactions under different in vitro conditions.

Mentions: In ergothioneine biosynthesis, the net-transfer of sulfur from cysteine to histidine side-chain is the combination of two reactions: a mononuclear non-heme iron catalyzed oxidative C-S bond formation (EgtB or Egt1 catalysis, Fig. 1) and a PLP-dependent C-S lyase (EgtE). Due to the lack of access to M. smegmatis EgtE enzyme, the proposed C-S lyase activity in M. smegmatis was not reconstituted in vitro. Studies were conducted using M. smegmatis cell lysate36. In this work, we have successfully produced EgtE protein and fully developed its catalytic system. Thus, for the first time, the ergothioneine biosynthetic pathway from M. smegmatis was fully reconstituted in vitro. EgtE enzymatic activities were characterized in vitro under a few different conditions: the C-S lyase activity for thio-ether 8 and the C-S lyase activities for sulfoxide 4 with and without reductants (Fig. 7). In all three reactions, pyruvate and ammonia were produced as the side-products. When thio-ether 8 was used as substrate, ergothioneine was the end product whether a reductant is present or not. However, when sulfoxide 4 was used as the substrate and in the absence of a reductant, thiol ester of thio-sulfinic acid (13) was the end product. Its presence was supported by the isolation of its hydrolyzed products: ergothioneine (5) and ergothioneine-2-sulfinic acid (14). When sulfoxide 4 was used as substrate and by including DTT as a reductant in the reaction mixture, only ergothioneine was produced. In the subsequent characterizations of EgtE catalysis using a coupled assay to monitor pyruvate formation rate, the kinetic parameters suggest that the sulfoxide 4 is the preferred substrate for EgtE enzyme. This is also consistent with previous studies on the mononuclear non-heme iron enzyme (Egt1, EgtB, or OvoA) catalyzed oxidative C-S bond formation reactions (Fig. 1)2021373875. Several lines of evidence suggest that sulfoxide 4 instead of a thio-ether 8 is the oxidative coupling product from these mononuclear non-heme iron enzyme catalyzed reactions: 1) H2O2 was not detected as a side-product in this reaction; 2) Thio-ether 8 was synthesized chemically and under the conditions, its oxidation to sulfoxide 4 by either O2 or H2O2 is below our detection limit; 3) When 40 × of catalase relative to OvoA, Egt1 was included in the reaction mixture, sulfoxide 4 was still the only detectable oxidative coupling product75. Thus, detailed biochemical characterizations of the two key enzymes (EgtB/Egt1 and EgtE) led to the full reconstitution of the ergothioneine biosynthetic pathway in vitro (Fig. 1).


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)

EgtE reactions under different in vitro conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: EgtE reactions under different in vitro conditions.
Mentions: In ergothioneine biosynthesis, the net-transfer of sulfur from cysteine to histidine side-chain is the combination of two reactions: a mononuclear non-heme iron catalyzed oxidative C-S bond formation (EgtB or Egt1 catalysis, Fig. 1) and a PLP-dependent C-S lyase (EgtE). Due to the lack of access to M. smegmatis EgtE enzyme, the proposed C-S lyase activity in M. smegmatis was not reconstituted in vitro. Studies were conducted using M. smegmatis cell lysate36. In this work, we have successfully produced EgtE protein and fully developed its catalytic system. Thus, for the first time, the ergothioneine biosynthetic pathway from M. smegmatis was fully reconstituted in vitro. EgtE enzymatic activities were characterized in vitro under a few different conditions: the C-S lyase activity for thio-ether 8 and the C-S lyase activities for sulfoxide 4 with and without reductants (Fig. 7). In all three reactions, pyruvate and ammonia were produced as the side-products. When thio-ether 8 was used as substrate, ergothioneine was the end product whether a reductant is present or not. However, when sulfoxide 4 was used as the substrate and in the absence of a reductant, thiol ester of thio-sulfinic acid (13) was the end product. Its presence was supported by the isolation of its hydrolyzed products: ergothioneine (5) and ergothioneine-2-sulfinic acid (14). When sulfoxide 4 was used as substrate and by including DTT as a reductant in the reaction mixture, only ergothioneine was produced. In the subsequent characterizations of EgtE catalysis using a coupled assay to monitor pyruvate formation rate, the kinetic parameters suggest that the sulfoxide 4 is the preferred substrate for EgtE enzyme. This is also consistent with previous studies on the mononuclear non-heme iron enzyme (Egt1, EgtB, or OvoA) catalyzed oxidative C-S bond formation reactions (Fig. 1)2021373875. Several lines of evidence suggest that sulfoxide 4 instead of a thio-ether 8 is the oxidative coupling product from these mononuclear non-heme iron enzyme catalyzed reactions: 1) H2O2 was not detected as a side-product in this reaction; 2) Thio-ether 8 was synthesized chemically and under the conditions, its oxidation to sulfoxide 4 by either O2 or H2O2 is below our detection limit; 3) When 40 × of catalase relative to OvoA, Egt1 was included in the reaction mixture, sulfoxide 4 was still the only detectable oxidative coupling product75. Thus, detailed biochemical characterizations of the two key enzymes (EgtB/Egt1 and EgtE) led to the full reconstitution of the ergothioneine biosynthetic pathway in vitro (Fig. 1).

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