Selenocysteine as a Substrate, an Inhibitor and a Mechanistic Probe for Bacterial and Fungal Iron-Dependent Sulfoxide Synthases.

Sulfoxide synthases are non-heme iron enzymes that participate in the biosynthesis of thiohistidines, such as ergothioneine and ovothiol A. The sulfoxide synthase EgtB from Chloracidobacterium thermophilum (CthEgtB) catalyzes oxidative coupling between the side chains of N-α-trimethyl histidine (TMH) and cysteine (Cys) in a reaction that entails complete reduction of molecular oxygen, carbon-sulfur (C-S) and sulfur-oxygen (S-O) bond formation as well as carbon-hydrogen (C-H) bond cleavage. In this report, we show that CthEgtB and other bacterial sulfoxide synthases cannot efficiently accept selenocysteine (SeCys) as a substrate in place of cysteine. In contrast, the sulfoxide synthase from the filamentous fungus Chaetomium thermophilum (CthEgt1) catalyzes C-S and C-Se bond formation at almost equal efficiency. We discuss evidence suggesting that this functional difference between bacterial and fungal sulfoxide synthases emerges from different modes of oxygen activation.

An Alternative Active Site Architecture for O2 Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum.

Sulfoxide synthases are nonheme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-α-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the γ-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system, we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.

Structure of the sulfoxide synthase EgtB from the ergothioneine biosynthetic pathway.

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.

Conversion of a non-heme iron-dependent sulfoxide synthase into a thiol dioxygenase by a single point mutation.

EgtB from Mycobacterium thermoresistibile catalyzes O2-dependent sulfur-carbon bond formation between the side chains of Nα-trimethyl histidine and γ-glutamyl cysteine as a central step in ergothioneine biosynthesis. A single point mutation converts this enzyme into a γ-glutamyl cysteine dioxygenase with an efficiency that rivals naturally evolved thiol dioxygenases.