Cytochromes P450 (CYP) are a superfamily of heme-containing monooxygenases that are versatile catalysts of a variety of anabolic and catabolic processes. These enzymes are found in all kingdoms of life and metabolize a broad range of substrates, including drugs, endogenous hormones and vitamins, and environmental pollutants. Of the 57 CYP enzymes found in the human body, a subset consisting mainly of subfamilies 1, 2, and 3 are involved in drug and xenobiotic metabolism. Our research focuses on how the physical structure of two subfamilies of xenobiotic metabolizing CYP enzymes, CYP2B and CYP3A, guides their catalytic function. A combination of solution biophysics, enzyme biochemistry, and structural biology is used to probe these structure-function relationships.
Structural Basis of CYP2B Function
This subfamily of CYP enzymes catalyzes the oxidation of a broad range of substrates that include the endogenous molecules testosterone and arachidonic acid, multiple pharmaceutically relevant drugs such as efavirenz and artemether, and environmental pollutants including polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs). The CYP2B enzymes show relatively low catalytic conservation across mammalian species and thus serve as a good platform for the study of enzyme structure-function relationships.
In order to probe the structural basis of CYP2B enzyme function, X-ray crystallography has been used extensively. Information from the available X-ray crystal structures provides plausible reasons for the ability of CYP2B enzymes to interact with a diverse set of chemicals from cyclohexane (Mr ≈ 84) and toluene (Mr ≈ 92) to mifepristone (Mr ≈ 429) and multiple molecules of amlodipine (Mr ≈ 409 × 2). Rearrangements of CYP2B proteins that accommodate the diversity of ligand interactions include movements of the F-G cassette, which is made up of the F-, F’-, G’-, and G-helices, the I-helix, and the B’-C loop region.
At the same time, the functional importance of various amino acid residues outside the enzyme active site or in a putative substrate access channel have been probed using steady-state kinetics and stoichiometric measurements of substrate oxidation. Alteration of residues in either of these regions were linked to changes in enzyme expression, stability, or function. Many single nucleotide polymorphisms (SNPs) found in human xenobiotic metabolizing CYP enzymes are found away from the enzyme active site. SNPs in human CYP enzymes have been linked to altered mRNA splicing, protein expression, and enzyme stability or function.
Cytochromes P450 from Wild Herbivores
Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds (PSCs), many of which have medicinal value or are drugs. Herbivores must cope with these toxins while consuming plant matter for sustenance, and some species such as Stephen’s woodrat are able to thrive on diets containing high concentrations of these toxins. After the laboratory of Denise Dearing at the University of Utah clonedcyp2b genes from Desert woodrats, our lab used a mix of classic biochemical and biophysical assays to characterize unique CYP enzymes from wild herbivores. The ability of these animals to cope with diets containing complex mixtures of potential toxins is partially due to small changes in protein sequence that have large effects on metabolism of substrates.
Molecular Basis of CYP3A4 Function
In humans CYP3A4 oxidizes more than 50% of all clinically relevant drugs. Oxidation of many of these substrates displays sigmoidal kinetics indicating allosteric interactions between the ligands and the enzyme. Thus, the molecular basis of CYP3A4 function and allostery is of great interest for pharmaceutical drug development. Conformational changes due to ligand binding and/or protein-protein interactions play key roles in the atypical (non-Michaelis Menten) kinetics of CYP3A4-catalyzed oxidations.
Using fluorescence resonance energy transfer (FRET) and a model substrate, heme spin-state changes upon ligand binding to CYP3A4 are linked to a high-affinity protein-ligand complex, and additional ligand binding events induce further conformational changes in the protein. However, evidence of single CYP enzymes existing in multiple conformations in solution is mounting with examples from bacteria, CYP119 (P450 eryK) and CYP261, and CYP2B4. Thus, the possibility that CYP3A4 exists in multiple conformations inside the cell is likely. Cooperative effects in ligand binding have been seen between the CYP enzyme and multiple molecules of the same ligand as well as the CYP enzyme and individual molecules of different ligands. Furthermore, experiments probing CYP3A4-CYP3A4 and CYP3A4-CYP2E1 protein-protein interactions indicate that allosteric effects on ligand binding or substrate turnover are dependent upon protein concentration within a membrane surface.