Structural insights into the convergent evolution of sulfoxide synthase EgtB-IV, an ergothioneine-biosynthetic homolog of ovothiol synthase OvoA

Non-heme iron-dependent sulfoxide/selenoxide synthases (NHISS) constitute a unique metalloenzyme class capable of installing a C–S/Se bond onto histidine to generate thio/selenoimidazole antioxidants, such as ergothioneine and ovothiol. These natural products are increasingly recognized for their health benefits. Among associated ergothioneine-biosynthetic enzymes, type IV EgtBs stand out, as they exhibit low sequence similarity with other EgtB subfamilies due to their recent divergence from the ovothiol-biosynthetic enzyme OvoA. Herein, we present crystal structures of two representative EgtB-IV enzymes, offering insights into the basis for this evolutionary convergence and enhancing our understanding of NHISS active site organization more broadly. The ability to interpret how key residues modulate substrate specificity and regioselectivity has implications for downstream identification of divergent reactivity within the NHISS family. To this end, we identify a previously unclassified clade of OvoA-like enzymes with a seemingly hybrid set of characteristics, suggesting they may represent an evolutionary intermediate between OvoA and EgtB-IV. [Display omitted] •Hercynine-bound structures offer insights into the convergent evolution of EgtB-IV•EgtB-IV cannot be converted into OvoA through simple active site mutations•An active site lid-loop dictates cysteine specificity in OvoA-like enzymes•A potential evolutionary intermediate between OvoA and EgtB-IV is presented Ireland et al. report X-ray crystal structures of type IV EgtB enzymes involved in ergothioneine biosynthesis. The results shed light on the convergent evolution of sulfoxide synthase EgtB-IV, which features closest homology to ovothiol-biosynthetic OvoA. Additionally, they clarify the structural basis for EgtB-IV’s specificity for cysteine as the S-donor.