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P450 Structure and Function

Cytochromes P450 are a group of enzymes so complex they have been termed 'diversozymes'. These heme containing proteins use molecular oxygen and electrons to catalyze the formation of many hormones and to detoxify xenobiotics (foreign substances, including pharmaceutical drugs, caffeine, nicotine, etc.) among other reactions. Though the oxidations they carry out are very diverse, the enzymes themselves share many conserved features and generally appear to operate through a common chemical mechanism.

At the left is a picture of the water in the active site of P450BM-3 when substrate is not bound. When substrate binds to a P450, the water in the center moves to the left and the heme iron spin state changes from low-spin to high-spin, making it much easier to reduce. This is one mechanism by which reducing equivalents can be protected. The crystal structure of P450BM-3 complexed to the substrate N-palmitoylglycine solved at UTSouthwestern in Bill Peterson's Lab and the Structural Biology Lab reveals that this is accomplished not by total displacement of the heme's axial water ligand but through a very subtle movement of this water to the groove in the highly conserved region of the I-helix.

In the background of this page you see the FMN and FAD cofactors of Cytochrome P450 Reductase as they can be found in the crystal structure 1AMO in the PDB database. Electrons are taken from nicotinamide cofactors and are transferred through FAD to FMN on the way to the P450. Not all P450s make use of this protein for their source of electrons, though many do.

Bacterial P450s As Catalysts

The reactions catalyzed by P450s makes them ideal catalysts for functionalization of small molecules. Although there are many specific reactions catalyzed by these enzymes, the most common are hydroxylation or epoxidation of alkenes. With the rapid increase in the number of microbial genomes solved, there are thousands of microbial P450s whose sequences are known but whose substrate specificity has not been characterized.

We are actively cloning and expressing these P450s to characterize their ability to functionalize small molecules like pharmaceutically interesting natural products. With many P450s in hand we can also explore structure and function in ways that were not accessible previously.

One particular focus is in the cloning and characterizing of enzymes in which the P450 portion is only one module of a multi-module enzyme. The historically studied CYP102A1, also called P450BM-3, is a great example. In this enzyme there is a P450 domain but there are also FAD-binding and FMN-binding domains that are involved in electron transfer. Bacteria seem to have found several interesting architectures like this one to make P450 catalysis more efficient.

Cholesterol 24S-Hydroxyase and Brain Cholesterol Metabolism

The brain is very rich in cholesterol, which is important for regulating the properties of neuronal membranes so that synaptic transmission can occur efficiently. The blood-brain barrier prevents cholesterol from entering or leaving the brain, resulting in a brain cholesterol pool that is largely separated from cholesterol metabolism in the rest of the body. Researchers identifying how cholesterol can leave the brain to be cleared by the liver discovered that a cytochrome P450 (CYP46A1) is responsible for hydroxylating the side chain of cholesterol to produce 24S-hydroxycholesterol, which can escape the blood brain barrier. Approximately 60% of cholesterol leaving the brain leaves by this route. Changes in this enzyme have been linked to a person's risk for Alzheimer's disease, the reaction at least in part regulated cholesterol synthesis in the brain, and there is recent evidence that it may play a role in higher order brain functions.

We conduct structure-function studies on the enzyme to understand the chemistry it carries out and the regulatory mechanisms that turn the reaction on and off. In research funded by the Welch Foundation, we also are mutating a highly efficient bacteria P450 to carry out this hydroxylation of cholesterol on a synthetic scale.

Lipid Signals: From Eicosanoids to Bacterial Quorum Sensing

The enzymes we study carry out a a variety of oxidations on saturated and unsaturated fatty acids that are often used in nature to generate interesting signals. Lipids make a great backbone for functionalization to incorporate elements like hydroxyl groups and epoxides that allow for a great deal of molecular recognition by other enzymes and receptors. Take for example, arachidonic acid. This single polyunsaturated fatty acid is converted to at least forty different signaling molecules known as eicosanoids, and the list grows continuously as new compounds are discovered.

Bacterial also use interesting lipid signals. One example we are examining in detail is a class of compounds known as acyl homoserine lactones, which are a fatty acid conjugated to an amino acid (homoserine is an amino acid, though not one of the twenty commonly used to build proteins). Acyl homoserine lactones are signals generated by some bacteria to control population dependent gene expression. This process is very important in symbiotic relationships, like the interaction of nitrogen fixing bacteria with roots of plants, and in pathogenic relationships like infection and dental plaque. We have discovered a potential role for P450 cytochromes in metabolism of acyl homoserine lactone signals. As we have learned about other quorum sensing signals, we have begun to study other lipid derived quorum sensing signals as well.

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This page is maintained by Dr. Donovan C. Haines.