Enzymatic strategies for carbon-sulfur bond formation in ergothioneine biosynthesis

Sulfur-containing molecules are abundant in nature and in pharmaceutical and agrochemical industries. Many chemical and enzymatic strategies for C-S bond formation have been identified and used to create novel compounds. Recently, there has been a considerable amount of interest in direct C-H bond functionalization, especially in reactions which have been developed to directly transform C-H to C-S bonds. Such transformations have also been observed in nature, for example, in ergothioneine biosynthesis. In Chapter 1, a brief overview on sulfur-containing molecules and synthetic/biosynthetic strategies to synthesise these is given. Ergothioneine is a sulfur-containing derivative of histidine with antioxidant properties. In the following sections of this thesis, a novel biosynthetic enzyme for direct C-H to C-S bond transformation is described; the anaerobic ergothioneine biosynthetic enzyme, EanB. The enzymes for oxygen-dependent ergothioneine biosynthesis (EgtA-E) were described a few years ago. In Chapter 3, the identification of an oxygen-independent ergothioneine biosynthetic pathway is described. The pathway involves only two enzymes - the methyl transferase EanA and the sulfurtransferase EanB. In addition to the in vitro reconstitution of oxygen-independent ergothioneine biosynthesis, we could show that the extremely halophilic bacterium, Salinibacter ruber, an organism carrying genetic information only for anaerobic ergothioneine biosynthesis, could produce ergothioneine in similar concentrations as has been described for those carrying the genetic information for aerobic ergothioneine biosynthesis. This study was followed up by a structural and mechanistic investigation of the EanB-catalyzed C-S bond formation, described in Chapter 4. Based on structural and kinetic data, a mechanistic model for the direct C-H to C-S bond transformation catalyzed by EanB was elucidated. EanB is suggested to follow a ping-pong mechanism where, in a first step, a persulfide is formed on an active site cysteine residue of EanB and, in a second step, a sulfane sulfur is transferred to the unactivated carbon 2 of the imidazole ring of N-α-trimethylhistidine. The use of a single turnover assay allowed us to investigate the sulfur transfer from the enzyme to the substrate isolated from the formation of the active site persulfide. In Chapter 5, the focus is set on the non-heme iron-dependent ergothioneine biosynthetic enzyme, EgtB. EgtB catalyzes the oxygen-dependent C-S bond formation in aerobic ergothioneine biosynthesis. We designed substrate analogs to probe the binding interactions between the two substrates, N-α-trimethyl histidine and cysteine/γ-glutamyl cysteine. The result of this study led to the development of bisubstrates which revealed the importance of substrate alignment for efficient C-S bond formation. Additionally, we analyzed substrate binding and regulation of the SAM-dependent methyl transferase EgtD, the only enzyme common to all known ergothioneine biosynthetic pathways. This study, described in Chapter 6, provides a good basis for further inhibitor design. This thesis gives an insight into two different strategies of C-S bond formation in ergothioneine biosynthesis. Our findings contribute to a further understanding of the role of ergothioneine in vivo and its molecular mechanism of action