The Saarlandes biochemistry team, led by Professor Bruce Morgan, then developed an assay to test the relevance of the mutations in live yeast cells, confirming the same pattern of results-all four mutations are required for complete transition between the two classes. "Mutations in all four areas work together to make the protein either an active redox catalyst or an iron-binding protein," Deponte said. Ultimately, they were able to turn inactive proteins, whose job is normally to sense or deliver iron-sulfur clusters, into active enzymes catalyzing redox reactions and vice versa. The researchers concluded that the long loop acts like an off switch in the inactive Class II, and that introduction of all four mutations from Class I glutaredoxins was required to fully convert the inactive protein into an active one. However, in combination with other mutations, the activity of the Class II protein progressively increased. When the researchers cut out the long loop and replaced it with the shorter one from a Class I protein, they observed slightly increased catalytic activity. The most notable physical difference between the two classes is a long loop present in the inactive Class II proteins. The team systematically replaced regions in the inactive Class II protein with corresponding regions from the active Class I proteins, and vice versa. Normally, an active Class I enzyme would catalyze, or assist, a redox reaction, which are chemical reactions involving the transfer of electrons between molecules. First, they produced and purified the proteins in order to analyze their activity in a test tube assay. To measure this, they used a combination of editing and tracking techniques. Deponte and his collaborators wanted to know exactly how much each mutation contributed to making the protein a catalytically active Class I or an inactive Class II glutaredoxin.Ĭomparison of the four determinant structural differences between enzymatically active and inactive glutaredoxins. Nuclear magnetic resonance structures revealed four areas containing differences, or mutations, between the Class I and Class II amino acid sequences. Deponte teamed up with researchers at the Universität des Saarlandes and Heinrich-Heine-Universität in Düsseldorf to investigate the proteins in test tubes, in yeast cells and in computational models. While biochemists have known about the two types for well over 20 years, it was unclear what structural differences are responsible for the distinct functions. Class II glutaredoxins are not active catalysts, but rather help sense and transport iron-sulfur clusters, a key part of iron metabolism. Class I glutaredoxins are enzymes that catalyze important redox reactions, such as the synthesis of the precursors of DNA. There are two major classes of glutaredoxin proteins. "Learning more about them improves our basic understanding of how life works." "These proteins are central to essential metabolic pathways," said Professor Marcel Deponte, a biochemist who led the research at the Technische Universität Kaiserslautern. Four mutations in a group of proteins, called glutaredoxins, determine how the proteins operate in everything from bacteria and yeast to plants and humans.
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