Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

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.

). Upon treatment with fluorophenylhydrazine, the pyruvate methyl group changed from solvent-exchangeable to non-exchangeable, which allowed us to directly quantify the ratio between ergothioneine and pyruvate from EgtE reaction mixture using 1 H-NMR directly. To quantitatively measure the ratio between ergothioneine 5 and the adduct 23, ethyl viologen was added as an internal standard to improve the accuracy of signal quantification.
Ethyl viologen has two signals in the 8 -9.5 ppm region (labeled as 2 and 3, colored green in the spectrum) and signals at  1.5 ppm region (labeled as 5 and colored green in the spectrum). The ratio between ergothioneine 5 and the ethyl viologen signals was calculated by measuring the ratio between ergothioneine 5 H-5 signal (labeled as 5 and colored red in the spectrum) and the ethyl viologen aromatic hydrogen signals. The ratio between the adduct 23 and the ethyl viologen signals was calculated by measuring the ratio between methyl group of the adduct 23 (labeled as 1 and colored blue in the spectrum) and the ethyl viologen ethyl group hydrogen signals (labeled as 5 and colored green in the spectrum). Based on the integration using ethyl viologen as an internal standard, the ratio of ergothioneine and pyruvate was  1: 1.

Supplementary Methods
Expression and purification of EgtE. The EgtE gene (GenBank: ABK70212.1) from Mycobacterium smegmatis str. MC2 155 was sub-cloned into the EcoRI and XhoI sites of pASK-IBA3+ expression vector from IBA GmbH.
The sequence of the recombinant EgtE is: The amino acids colored in red are extra amino acids introduced during the sub-cloning process. The amino acid sequence colored in blue is the Strep-tag, which is used for affinity-based purification by Strep-Tactin resin.
The EgtE-pASK-IBA3 + construct was transformed into BL21(DE3) cell and grown in4L LBmedium and DNase I (100 U/g cell) were then added into the cell suspension and the mixture was incubated on ice for 40 min with gentle agitation. The cells were disrupted by sonication (20 cycles of 30 s bursts). The supernatant and the cell debris were separated by centrifugation at 4 o C for 10 min at 20,000 g. To the supernatant (50 mL), streptomycin sulfate was added to a final concentration of 1% (w/v %) and the mixture was incubated on ice for 30 min with gentle agitation.
The white DNA precipitate was then separated by centrifugation at 20,000 g for 30 min at 4 C. The resulting supernatant was mixed with the Strep-Tactin resin (10 mL) and incubated on ice for 30 min. After the cell lysate was drained by gravity, the column was washed with washing buffer (100 mM Tris-HCl, 50 mM NaCl, pH 7.5) until the OD 260 is lower than 0.05. The recombinant protein was eluted with the elution buffer (2.5 mM desthiobiotin in 100 mM Tris-HCl buffer, pH 7.5). After the protein was concentrated by ultrafiltration, it was flash frozen by liquid nitrogen and stored at -80 C.
The typical yield is  2 mg of purified EgtE per gram of wet cells.