Examining the Mechanism of Egt2 in Ergothioneine Biosynthesis

Abstract

Ergothioneine (EGT), a unique histidine thiol‐derivative synthesized by certain species of bacteria and fungi, has been recognized as a powerful antioxidant that can have highly beneficial effects on human health. Ergothioneine's limited availability and potential benefits have spurred a great need to develop an efficient industrial scale production, which can best be achieved by engineering the bacterial/fungal production pathway. Interestingly, both of these pathways that produce EGT contain a terminal step in which a pyridoxal 5′‐phosphate (PLP)‐dependent C‐S lyase, termed Egt2, cleaves a carbon‐sulfoxide bond to produce EGT and pyruvate. In this study, we aimed to understand the mechanism of a novel C‐S lyase that uses sulfoxide as its substrate through a structure‐guided approach. In order to overcome a high turn‐over rate and capture the enzyme substrate complex during various stages of the reaction, we employed site‐directed mutagenesis (SDM) to mutate residues interacting with the co‐factor, PLP. Through SDM, we inserted mutations that compromised the enzyme's catalytic rate while preserving structural integrity. The crystallographic analyses revealed the structure of a highly reactive sulfenic acid intermediate that has not previously been visualized in protein chemistry.Support or Funding InformationAmerican Society of Biochemistry and Molecular Biology (ASBMB) & The University of Texas at Austin ‐ Senate of College Councils