Research
Research
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Image of human mitochondrial P450 11B2 showing a typical P450 fold with protein in blue, heme in red, and a steroid ligand in yellow (PDB ID: 4DVQ).
The human cytochrome P450 (P450) enzymes are monooxygenases involved in the metabolism of steroid hormones, pharmaceuticals, carcinogens, and fat-soluble vitamins. P450 enzymes therefore have many direct impacts on human health. The study of P450 enzymes has led to several important advances in medicine including new therapies and the safer use of existing therapies. Our work is currently focused on the human mitochondrial enzyme P450 27A1, which plays roles in both cholesterol metabolism and vitamin D metabolism. Therefore, learning more about how this enzyme functions could lead to important discoveries related to cholesterol metabolite signaling (such as with some cancers) and bone health.
Reactions catalyzed by human cytochrome P450 27A1.
In particular, we are interested in how the interactions between the P450 enzymes and the redox partner protein adrenodoxin can alter enzyme structure and in turn P450 function. It is well understood that human mitochondrial P450 enzymes must receive electrons from adrenodoxin. A growing body of evidence suggests that the interactions between the P450 enzyme and adrenodoxin affect more than just electron injection. These interactions also seem to be able to affect substrate binding and reaction kinetics. We assert that by better understanding all the ways in which adrenodoxin affects P450 catalysis, new ways of modifying P450 enzymes for clinical purposes can be developed. Additionally, what we learn about human mitochondrial P450 enzymes and their interactions with adrenodoxin may lead to important lessons about other P450 enzymes and their redox partner proteins. This could have impacts outside of medicine as P450 enzymes are also being explored as catalysts to make industrial processes more efficient and environmentally friendly.
Left, Example SDS-PAGE result of some proteins purified in the Reddish lab at Appalachian State. Lane 1 shows molecular weight standards. Lane 2 shows purified human adrenodoxin (Mr = 14 kDa). Lane 3 shows purified P450 27A1 (Mr = 58 kDa). Right, reduced CO-binding difference spectrum of the purified P450 27A1 indicating activity.
To examine P450 enzymes we use a variety biochemical and biophysical techniques. We typically use recombinant protein expression of human proteins in E. coli to gain access to the proteins we study. With our proteins available, we specialize in the use of steady state and presteady state kinetics to learn more about P450 mechanisms. We use spectroscopic and analytical techniques to observe these reactions, including UV-VIS absorbance, fluorescence, and HPLC-UV-MS. We also partner with other labs to study protein structure. Students in the Reddish lab get exposure to all of these techniques and gain experience with more techniques the longer they stay with the lab.
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