Ergothioneine is a histidine-derived thiol of bacterial and fungal origin that has also been isolated from animal and human tissue. Recent findings point to critical functions of ergothioneine in human physiology, but its role in microbial life is poorly understood. This report describes the identification of the ergothioneine biosynthetic gene cluster from mycobacteria and in vitro reconstitution of this process using recombinant proteins from Mycobacterium smegmatis. The key reactions are catalyzed by a methyltransferase that transfers three methyl groups to the alpha-amino moiety of histidine and an iron(II)-dependent enzyme that catalyzes oxidative sulfurization of trimethylhistidine. A search for homologous genes indicated that ergothioneine production is a frequent trait among fungi, actinobacteria, and cyanobacteria but also occurs in numerous bacteroidetes and proteobacteria.

Supplementary Methods General information All methyltransferase-encoding pET28a expression plasmids were purchased from BioCat GmbH. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions. The gels were stained with Coomassie brilliant blue. Analytical high-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20AT HPLC with a Shimadzu SPD-M20A diode array detector. Phenomenex Luna ® 5 µm SCX 100 Å, LC Column 100 x 4.6 mm was used for cation exchange. Size exclusion chromatography was performed on AKTA Explorer 100 FPLC System with a HiLoad ® 16/600 Superdex ® 200 pg column. High-resolution mass spectra were obtained on a Bruker maXis 4G UHR-TOF Mass Spectrometer. Chiral HPLC-UV analysis was performed on Agilent 1100 system with CHIRAL-AGP column from Chrom Tech, Inc. 1 H NMR spectra were recorded on a a Bruker Avance Neo NMR spectrometer operating at 500 MHz proton frequency and chemical shifts were internally referenced to residual proton signals of solvents. Chemical shifts (δ) were reported in parts per million (ppm). Standard abbreviations indicating multiplicity were used as follows: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), and m (multiplet). Coupling constants (J) were reported in Hertz (Hz). Unless otherwise noted, all chemicals and reagents were purchased from Sigma Aldrich and used as recieved. Antibiotics were purchased from PanReac AppliChem. Ingredients for buffers were purchased from Acros Organics. L-Histidine was purchased Fluka TM. Myo-inositol was purchased from Apollo Scientific ltd. 2,7-dihydroxynaphthalene was purchased from FluoroChem Co. Deuterated solvents were purchased from Cambridge Isotope Laboratories. Protein expression E. coli BL21(DE3) pLysE cell: pET-28a plasmids containing genes were transformed using standard heat-shock protocols for chemically competent E. coli BL21(DE3) cells. E. coli cells containing the plasmid were collected from LB-AGAR plates with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml) and used to inoculate LB medium with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml) (20 ml). After incubation at 37°C overnight, 15 ml of pre-culture was used to inoculate fresh LB Medium (1L) with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml). 1 The cells were grown in 3L shaking flask at 37 °C (170 rpm) for about 4 h until OD600 reached 0.6~0.7 and then the culture was cool to 18 °C and Isopropyl β-D-thiogalactoside (IPTG) was added to final concentration of 100 µM. Expression was performed for 20 hours. Cells were harvested by centrifugation at 10,000 x g for 30 minutes and stored at-20 °C.

The non-heme iron enzyme EgtB catalyzes O2 -dependent C-S bond formation between γ-glutamyl cysteine and N-α-trimethyl histidine as the central step in ergothioneine biosynthesis. Both, the catalytic activity and the architecture of EgtB are distinct from known sulfur transferases or thiol dioxygenases. The crystal structure of EgtB from Mycobacterium thermoresistibile in complex with γ-glutamyl cysteine and N-α-trimethyl histidine reveals that the two substrates and three histidine residues serve as ligands in an octahedral iron binding site. This active site geometry is consistent with a catalytic mechanism in which C-S bond formation is initiated by an iron(III)-complexed thiyl radical attacking the imidazole ring of N-α-trimethyl histidine.

Ovothiols are histidine-derived thiols that were first isolated from marine invertebrates. We have identified a 5-histidylcysteine sulfoxide synthase (OvoA) as the first ovothiol biosynthetic enzyme and characterized OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi . Homologous enzymes are encoded in more than 80 genomes ranging from proteobacteria to animalia.