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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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Enzymatic and chemical syntheses of EgtE substrate candidates (4 & 8).
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f2: Enzymatic and chemical syntheses of EgtE substrate candidates (4 & 8).

Mentions: When the M. smegmatis ergothioneine biosynthetic gene cluster was discovered, the in vitro activities of EgtA, EgtB, EgtC, and EgtD enzymes were verified. However, the proposed C-S lyase activity (EgtE) was not demonstrated in vitro due to difficulties in M. smegmatis EgtE overexpression. In this study, we report the isolation of EgtE protein and the detailed biochemical characterization of its novel C-S lyase activity. EgtE makes use of both sulfoxide (4) and thio-ether (8, Fig. 2) as substrates. In addition, different outcomes were observed when sulfoxide (4) was used as the substrate in the absence or in the presence of reductants. More in-depth kinetic characterizations suggest that sulfoxide 4 is the biological EgtE substrate. More importantly, subsequent studies led to the trap of a sulfenic acid intermediate in EgtE-catalysis. Small molecular sulfenic species are highly reactive in nature and stable small molecular sulfenic species are rare. The unique ergothioneine chemical property may stabilize the sulfenic species and explains our successful trapping of this species in EgtE reaction.


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)

Enzymatic and chemical syntheses of EgtE substrate candidates (4 & 8).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Enzymatic and chemical syntheses of EgtE substrate candidates (4 & 8).
Mentions: When the M. smegmatis ergothioneine biosynthetic gene cluster was discovered, the in vitro activities of EgtA, EgtB, EgtC, and EgtD enzymes were verified. However, the proposed C-S lyase activity (EgtE) was not demonstrated in vitro due to difficulties in M. smegmatis EgtE overexpression. In this study, we report the isolation of EgtE protein and the detailed biochemical characterization of its novel C-S lyase activity. EgtE makes use of both sulfoxide (4) and thio-ether (8, Fig. 2) as substrates. In addition, different outcomes were observed when sulfoxide (4) was used as the substrate in the absence or in the presence of reductants. More in-depth kinetic characterizations suggest that sulfoxide 4 is the biological EgtE substrate. More importantly, subsequent studies led to the trap of a sulfenic acid intermediate in EgtE-catalysis. Small molecular sulfenic species are highly reactive in nature and stable small molecular sulfenic species are rare. The unique ergothioneine chemical property may stabilize the sulfenic species and explains our successful trapping of this species in EgtE reaction.

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