Exploring New Structural and Functional Space in the Glutathione Transferase Superfamily from Escherichia coli K-12

Abstract

Genome sequencing projects have revealed that glutathione (GSH) transferases are widely distributed in bacteria but most remain only as annotations in sequenced genomes. The goal of this project was to assign function to members of the GSH transferase superfamily from Escherichia coli K-12. Several strategies are required for the accurate assignment of function to proteins of unknown function. These approaches include; 1) analysis using informatics and sequence similarity, 2) co-localization of genes providing operon/metabolic context, 3) transcriptional analysis, 4) phenotypic response to gene knockouts, 5) structural biology, 6) and functional assays of the protein. The work described in this report focuses on functional and structural studies of three GSH transferase homologs from E. coli, YfcG, YghU and YqjG. The YghU and YfcG proteins represent a previously unrecognized class of GSH transferases that have unique structural and catalytic properties. These two proteins have a distinct active site and the ability to bind glutathione disulfide (GSSG) with high affinity (YfcG) or two molecules of GSH simultaneously (YghU). The proteins share robust disulfide-bond oxidoreductase and GSH-dependent peroxidase activity. Despite the similarities, YghU and YfcG have significant chemical and structural differences, including their preferred oxidation states and the N-terminal and C-terminal extensions of YghU. YqjG belongs to a unique cluster of GSH transferases that has not been previously characterized. The protein exhibits modest disulfide bond reductase activity and may have glutathionyl-hydroquinone reductase activity with other substrates. The protein adopts a classical GSH transferase fold but a unique splayed dimer. Proteins in this class are primarily from bacteria, although some are from eukaryotes including fungi, and may constitute a novel class of GSH transferases.