Radical SAM Enzymes: Molecular Mechanisms of Radical Initiation

琼·布罗德里克。

Broderick, Joan B. 5R35GM131889-02 Administrative Supplement for Purchase of a Rapid Freeze Quench / Stopped Flow Abstract/Summary of Project Radical SAM is one of the largest enzyme superfamilies known, with its members catalyzing a remarkable diversity of reactions in all domains of life. Radical SAM enzymes are involved in the synthesis of essential cofactors and antibiotics, repair of DNA damage, and the assembly of complex biological metal clusters, among many other reactions. The presence of radical SAM enzymes in humans as well as in both beneficial and pathogenic microbes lends high significance to understanding their fundamental properties and mechanisms. The proposed research will develop a critical understanding of radical SAM mechanisms, in particular the radical initiation process common to all enzymes in this large and diverse superfamily. Research efforts will focus on trapping radical intermediates using rapid freeze-quench, cryoreduction, annealing, and photolysis approaches. These intermediates will then be characterized by using spectroscopic approaches such as electron paramagnetic resonance and electron-nuclear double resonance, and structural approaches such as X-ray crystallography. The proposed supplement will allow the purchase of needed stopped-flow mixing/ rapid-freeze-quench and UV-visible detection capability to carry out these experiments..