The catalytic mechanism of the iron-dependent sulfoxide synthase EgtB

Sulfoxide synthases EgtB form a class of non-haem iron enzymes, which catalyze the oxygen-dependent sulfur-carbon bond formation between low molecular weight thiols and Nα,Nα,Nα-trimethyl-L-histidine as a central step in ergothioneine biosynthesis. The crystal structure of EgtB from Mycobacterium thermoresistibile, in complex with γ-glutamylcysteine and Nα,Nα-dimethyl-L-histidine, implicate both substrates and three histidine residues as ligands in an octahedral iron binding site. In the secondary coordination sphere we identified a tyrosine residue which serves as a proton donor to an iron(III)-superoxo species. Mutation of this residue to phenylalanine produced a variant with 500-fold reduced sulfoxide synthase activity. Moreover, this protein catalyzes thiol dioxygenation with an efficacy that rivals naturally evolved cysteine dioxygenases. We also demonstrated that a catalytic tyrosine residue is present among different sulfoxide synthases.
Furthermore, the replacement of cysteine with selenocysteine in EgtB from Candidatus chloracidobacterium thermophilum B might catalyze the formation of the selenoxide, which is further reduced to hercynylselenocysteine. We suggest that the enzymes involved in the biosynthetic pathway of ergothioneine are able to synthesize selenoneine, where first the selenoxide is formed by the sulfoxide synthase EgtB, which is then reduced by the intracellular reductants, and then the β-lyase EgtE catalyzes selenoneine formation. However, the enzymatic formation of the C-Se bond has a moderate rate in comparison to C-S bond formation. Additionally, selenocysteine is an excellent mechanistic probe; it acts as a competitive inhibitor towards cysteine and uncompetitive towards TMH, suggesting a sequential binding order in the mechanism of EgtB.
Protein design based on the crystal structure of EgtB from Mycobacterium thermoresistibile allowed the remodeling of the active site and the tuning of the reactivity of the sulfoxide synthase by introducing an additional hydrogen bond to the thiolate coordinated to the iron center of the enzyme. It was found that the resulting hydrogen bond between the thiolate of the substrate and S82 in the active site disturbs the formation of the proposed thiyl radical. This intermediate is required in the catalytic mechanism to further proceed to attack of this thiyl radical on the imidazole ring of the second substrate.
Overall we have used crystallographic data and kinetic analysis to probe the mechanistic details of EgtB-catalyzed C-S bond formation. This data would allow us to probe the activity of related enzymes as well as designing antibacterial inhibitors.