The Diversity of the Iron-dependent Sulfoxide Synthases in Ergothioneine Biosynthesis

The understanding of any biological system must start with a detailed understanding of the chemistry of the individual enzymes, their catalytic mechanisms and their structures. Ergothioneine, a small molecular weight thiol, is an ideal target for molecular dissection, due its ubiquity in life and complex and enigmatic physiological function. EgtB, an iron dependent sulfoxide synthase, was discovered through this approach, and represents an entirely new catalyst type, that is distinct in both reactivity and structure from other iron oxygenases. This makes dissection of its mechanistic details an attractive endeavor. As EgtB is also the central and characteristic enzyme of the oxidative pathway for ergothioneine biosynthesis, exploration of its reactivity and evolutionary history may shed light on questions pertaining to the emergence of ergothioneine.
EgtB catalyzes oxidative carbon sulfur bond formation between the C2 carbon of N,N,N-a-Trimethyl-histidine and the sulfhydryl group of cysteine or g-glutamyl-cysteine to form a hercynine-(g-glutamyl)cysteine-sulfoxide conjugate in a four electron oxidation that is coupled to the reduction of molecule oxygen to water. Despite having been the focus of numerous biochemical, bioinformatic and computational studies, the evolutionary history and catalytic mechanism of EgtB are respectively unknown and disputed.
This thesis tackles both questions through an approach which involves the characterization of divergent EgtB homologues. Crystal structures of these homologues, compounded with kinetic and/or bioinformatic characterization revealed that the EgtB family is characterized by extreme active site diversity. This diversity manifests itself in changes in catalytic residues, substrates and perhaps even reactivity, all of which can be assigned to a particular sequence motif. These differences provide a platform to explore the mechanism of EgtB via comparative enzymology, and allowed us to explore possible evolutionary routes to the diverse EgtBs. The crystal structures alone provide a valuable test of any mechanistic proposal. We envisage this work will drive mechanistic discussions and further exploration of the EgtB sequence space to capture the full diversity of this family and consequently ergothioneine biosynthesis.
The same strategy, combining bioinformatic analysis, in vitro characterization and structural biology, was leveraged in the identification and characterization of new enzymes and proteins that catalyze and are involved in other aspects of ergothioneine metabolism. These examples highlight how unravelling the molecular basis of ergothioneine provides insights into the biology and importance of this molecule.