Large-Scale Determination of Sequence, Structure, and Function Relationships in Cytosolic Glutathione Transferases across the Biosphere

The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu). Author Summary Cytosolic glutathione transferases (cytGSTs) are a large and diverse superfamily of enzymes that have important roles in metabolism and defense against oxidative damage. They have been studied for several decades but because of the synthetic nature of the chemicals used to test these proteins to determine if they have cytGST activity, little is known about the physiological reactions and roles of cytGSTs. In this large, collaborative study, we constructed networks where more than 13,000 cytGST sequences were grouped by sequence similarity and then used these networks to prioritize new targets for experimental characterization in relatively unexplored regions of the superfamily. We report here experimental results confirming GST-like activity for 82 of them, along with 37 new three-dimensional molecular structures determined for 27 targets. These new data, along with experimental data previously reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and illuminate the complex ways in which their variations in sequence and structure affect our ability to predict unknown functional properties.