To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH ( Cytochromes P4503 of the 1, 2, and 3 families play a central role in drug metabolism and detoxification (1). Unlike most classical enzymes with their strict substrate selectivity, these microsomal P450s can each bind and metabolize a number of substrates that are different in size, shape, and stereochemistry. A challenging problem is to understand how microsomal P450s accommodate different substrates and oxidize each in a regiospecific and stereo-specific manner. At the tertiary structural level, P450s exhibit a similar overall fold with a well conserved heme-binding core (2). However, recent results demonstrate that microsomal P450s exhibit striking conformational diversity and plasticity (3-5). Not only do different P450s exhibit different conformations, but an individual P450 can adopt multiple conformations in response to various ligands, making it difficult to model protein-ligand interactions even when one atomic structure of a P450 is known. Therefore, a good sampling of P450 "conformational space" is a prerequisite for understanding P450-ligand interactions and for performing modeling studies with high predictive power.P450s from the 2B subfamily are inducible by barbiturates and have been the object of numerous biochemical and biophysical investigations for several decades. P450 2B4, in particular, was the first microsomal P450 to be purified and is one of the best characterized xenobiotic metabolizing P450s (6). Recent structural investigations using an N-terminal modified form of P450 2B4 (2B4dH) have revealed that the enzyme can adopt strikingly different conformations (4, 5), making it one of the best sources for study of P450 conformational plasticity. In addition, a wealth of site-directed mutagenesis data on 2B4 and other 2B enzymes are available to correlate structural features with functional properties (7). When P450 2B4dH was crystallized without ligand, a tight dimer was formed by two symmetrical molecules that each adopted a widely open conformation (4). A large cleft is formed by helices F through G on one side and the BЈ/C loop and helix C on the other side (Fig. 1A). The cleft is ϳ15 Å wide and is filled by helices FЈ and GЈ of the symmetry-related molecule, whose His-226 forms an intermolecular coordinate bond to the heme iron of the other monomer. In order to investigate the structure of 2B4 with a ligand bound, the H226Y mutant was created to minimize dimer formation (5). When 2B4dH(H226Y) was crystallized with the inhibitor 4-(4-chlorophenyl)imidazole (CPI), a closed conformation was observed (Fig. 1B), which resembles those of P450s 2C5 (8 -10), 2C8 (11), 2C9 (12, 13), and 3A4 (14, 15). The BЈ/C loop and the N terminus of helix I move toward the active site, making contact with CPI. Helices F through G m...

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Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed ϳ3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu 264 3 Phe substitution did not alter the K s for inhibitors or the substrate benzphetamine, the K m for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.For decades, cytochromes P450 from the 2B subfamily have served as a prototype for investigation of the mechanism by which P450s metabolize substrates, especially drugs and environmental contaminants, of various size, shape, and polarity. Structure-function relationships of these enzymes have been studied extensively using chimeragenesis, sitedirected and random mutagenesis, molecular modeling, X-ray crystallography, and solution biophysics (Domanski and Halpert, 2001;Zhao and Halpert, 2007). X-ray structures of an engineered rabbit P450 2B4dH (N-terminal modified and C-terminal His-tag) in ligand-free (pdb code 1PO5), 4-(4-chlorophenyl)imidazole (4-CPI)-bound (pdb code 1SUO), and bifonazole (BIF)-bound (pdb code 2BDM) forms have provided structural evidence that the enzyme can undergo large ligand-induced conformational changes while maintaining its overall fold (Scott et al., 2003(Scott et al., , 2004Zhao et al., 2006). Subsequently, solution thermodynamic studies using isothermal titration calorimetry (ITC) with several imidazole inhibitors of different sizes showed the flexibility of P450 2B4 in accommodating a variety of ligands . These studies provide important insight into factors that must be considered...

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 Å 3 in the 4-CPI complex to 280 Å 3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine tuning of residue orientations and thermodynamic signatures.Cytochromes P450 of the 1, 2, and 3 families play a central role in drug metabolism and detoxification (1). Many P450s display broad and overlapping substrate specificities, yet an individual P450 often oxidizes a particular substrate with striking regio-and/or stereoselectivity. The importance of P450s in healthcare has led to an intense interest in developing

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

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Iron release process of ovotransferrin N-lobe (N-oTf) to anion/chelators has been resolved using kinetic and mechanistic approach. The iron release kinetics of N-oTf were measured at the endosomal pH of 5.6 with three different anions such as nitrilotriacetate, pyrophosphate, and sulfate using stopped flow spectrofluorimetric method, all yielding clear biphasic progress curves. The two observed rate constants and the corresponding amplitudes obtained from the double exponential curve fit to the biphasic curves varied depending on the type and concentration of anions. Several possible models for the iron release kinetic mechanism were examined on the basis of a newly introduced quantitative equation. Results from the curve fitting analyses were consistent with a dual pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first order rate constants of K 1 and K 2 (X, domain closed holo N-oTf; Y, anion induced different conformer of holo N-oTf). The reversible interconversions of X to Y and Y to X are driven by the second order rate constant k 3 and the first order rate constant K 4 , respectively. The obtained rate constants were greatly variable for the three anions depending on the synergistic or nonsynergistic nature. In the light of the anion-binding sites of N-oTf located crystallographically, the compatible mechanistic model that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.