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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.


Related in: MedlinePlus

UV-visible spectra of EgtE (30 μM) at a few different pHs.
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f3: UV-visible spectra of EgtE (30 μM) at a few different pHs.

Mentions: We sub-cloned M. smegmatis EgtE gene into the pASK-IBA3+ vector and the protein was overexpressed in the E. coli BL21(DE3) strain. The expressed EgtE was then purified using Strep-Tactin resin to near homogeneity (higher than 95% based on the analysis of the SDS-PAGE, supplementary Fig. 1). Furthermore, as suggested by EgtE sequence analysis, its UV-visible spectra are consistent with the presence of a PLP cofactor (Fig. 3). The pH-dependence of the EgtE internal aldimine electronic absorption was also studied. In PLP-containing proteins, the PLP-Lysine conjugate can exist in a few different forms: enolimine (9), ketoenamine (10) or deprotonated ketoenamine (11)2425262728293031. For the isolated EgtE protein at pH 7.5, it has an absorption feature near 420 nm (Supplementary Fig. 1), which is consistent with the presence of ketoenamine (10) tautomer of the protonated Schiff base. As the pH changes from 6.0 to 10.0, the UV-vis spectra (Fig. 3) do not show distinct changes in the overall absorption features and the maximal absorption wavelengths. Thus, for EgtE, the internal aldimine is present mainly in the form of 10. The amount of PLP per EgtE monomer was also determined after it was released by 0.2 M NaOH treatment32. The isolated EgtE has 0.8 mole PLP per mole of EgtE monomer.


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)

UV-visible spectra of EgtE (30 μM) at a few different pHs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: UV-visible spectra of EgtE (30 μM) at a few different pHs.
Mentions: We sub-cloned M. smegmatis EgtE gene into the pASK-IBA3+ vector and the protein was overexpressed in the E. coli BL21(DE3) strain. The expressed EgtE was then purified using Strep-Tactin resin to near homogeneity (higher than 95% based on the analysis of the SDS-PAGE, supplementary Fig. 1). Furthermore, as suggested by EgtE sequence analysis, its UV-visible spectra are consistent with the presence of a PLP cofactor (Fig. 3). The pH-dependence of the EgtE internal aldimine electronic absorption was also studied. In PLP-containing proteins, the PLP-Lysine conjugate can exist in a few different forms: enolimine (9), ketoenamine (10) or deprotonated ketoenamine (11)2425262728293031. For the isolated EgtE protein at pH 7.5, it has an absorption feature near 420 nm (Supplementary Fig. 1), which is consistent with the presence of ketoenamine (10) tautomer of the protonated Schiff base. As the pH changes from 6.0 to 10.0, the UV-vis spectra (Fig. 3) do not show distinct changes in the overall absorption features and the maximal absorption wavelengths. Thus, for EgtE, the internal aldimine is present mainly in the form of 10. The amount of PLP per EgtE monomer was also determined after it was released by 0.2 M NaOH treatment32. The isolated EgtE has 0.8 mole PLP per mole of EgtE monomer.

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