Professor Ken Houk and Dr Jacob Sanders in his group have provided computational evidence to support a new mechanism of action for FAD.
Flavin adenine dinucleotide (FAD) is a well-known enzymatic cofactor that catalyzes a variety of oxidative transformations in biochemical pathways.
The research, published in the Proceedings of the National Academy of Sciences (PNAS), was performed in collaboration with Professor Robin Teufel at the University of Freiburg in Germany as well as other scientists at the University of Michigan and the Scripps Institution of Oceanography. State-of-the-art quantum mechanical computations to support this new mechanism of action were performed by Dr. Sanders, a National Institutes of Health (NIH) Postdoctoral Fellow in Houk’s group (pictured right).
One common type of transformation catalyzed by FAD is the introduction of an oxygen atom into a substrate molecule. These transformations include the oxidation of an alkene to form an epoxide or the functionalization of a C-H bond to form an alcohol. In nearly all known cases, these transformations proceed through a reaction between FAD and molecular oxygen to form a flavin-C4a-hydroperoxide; subsequently one of the oxygen atoms from the hydroperoxide is transferred to the substrate. However, in tour de force X-ray crystallography experiments, Teufel and coworkers succeeded in crystallizing an enzyme known as EncM with molecular oxygen in the active site. The crystal structure, shown below, shows that molecular oxygen is not likely to bind in the usual orientation to form the flavin-C4a-hydroperoxide; rather, the position of bound molecular oxygen found in the structure suggests that oxygen is poised to form a previously unknown flavin-N5-oxide, after loss of water
State-of-the-art quantum mechanical computations performed in the Houk group confirmed that the formation of the flavin-N5-oxide was energetically feasible. Moreover, the computations revealed that the positioning of molecular oxygen in the EncM crystal structure closely matched the ideal geometry of the transition state leading to flavin-N5-oxide formation, demonstrating that the enzyme active site evolved to mimic and stabilize this critical transition state. In addition to revealing a new mode of action for a cofactor that is ubiquitous throughout metabolism, this work also demonstrates how enzymes can tune the reactivity of molecular oxygen and provides critical knowledge for future rational design of oxidative enzymes.
To learn more about the Houk group’s research, visit their website.
Figure 1: This figure illustrates the similarity between the EncM crystal structure with O2 bound (left) and the ideal transition state geometry (right) for the formation of flavin-N5-oxide, revealing how the enzyme has evolved to mimic and stabilize this transition state.
Figure 2: The structure of the well-established flavin-C4a-hydroperoxide (left) and flavin-N5-oxide (right) proposed to be an intermediate in EncM-catalyzed reactions.
