Biomedical subjects

James R Halpert

Publications and source records attributed to James R Halpert.

At least 19 recordsLinked to original sources

PubMed2007

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.

2-Naphthylamine↗

PubMed2006

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide.

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.

Animals↗

PubMed2006

Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme.

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.

Amino Acid Substitution↗

PubMed2006

Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

Benzene Derivatives↗

PubMed2006

Structure-function analysis of cytochromes P450 2B.

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure-function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.

Animals↗

PubMed2006

Variable path length and counter-flow continuous variation methods for the study of the formation of high-affinity complexes by absorbance spectroscopy. An application to the studies of substrate binding in cytochrome P450.

Studies of the equilibrium of protein-ligand interactions and determination of the stoichiometry of protein complexes constitute an important element of routine biochemical practice. In this paper we describe two innovative modifications of Job's method of continuous variation, which allow us to analyze tight interactions and determine stoichiometry in multi-site binding systems, including cases where the absorbance of the ligand overlaps with that of the enzyme-ligand complex. Our results on the interactions of cytochromes P450 3A4 and P450eryF with substrates illustrate the applicability of these approaches to the studies of substrate binding in enzymes that exhibit homotropic cooperativity.

Binding Sites↗

PubMed2006

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies.

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.

Amino Acid Sequence↗

PubMed2006

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration.

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.

Binding Sites↗

PubMed2006

Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains. Solution thermodynamics and molecular modeling.

Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 microM, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 microM and following the same rank order as the IC50 values. Changes in enthalpy at 25 degrees C ranged from -6.5 to -8.8 kcal mol(-1). The largest difference in binding entropy (+5.9 versus -4.1 cal mol(-1) K(-1)) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol(-1) K(-1)), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4.

Acrylamides↗

PubMed2005

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction.

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(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.

Antifungal Agents↗

PubMed2005

Kinetics of dithionite-dependent reduction of cytochrome P450 3A4: heterogeneity of the enzyme caused by its oligomerization.

To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.

Biopolymers↗

PubMed2005

Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts.

Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.

Animals↗

PubMed2005

Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.

The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.

Allosteric Regulation↗

PubMed2005

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe.

To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.

Allosteric Site↗

PubMed2005

Possible pathway(s) of testosterone egress from the active site of cytochrome P450 2B1: a steered molecular dynamics simulation.

To probe the possible substrate exit channel(s) in cytochrome P450 (P450) 2B1 and to clarify the role of residues previously identified by site-directed mutagenesis, a homology model was constructed based on the X-ray crystal structure of a P450 2B4-inhibitor complex. Testosterone was docked into the active site of P450 2B1 and was then pulled out through three putative channels using steered molecular dynamics simulations. The results indicated that of the three channels, the "solvent channel," lined by helices E, F, and I and the beta3 hairpin, required the largest rupture force and backbone motion, which rendered it unlikely as an exit route. The relatively small rupture forces and backbone motions for the other two channels suggested them as possible candidates for testosterone passage. The opening of channel 1, located between helices G and I and the B'-C loop, is characterized by rotation of the aromatic ring of Phe297 together with a bending of the B'-C loop. The opening of channel 2, penetrating through the B'-C loop/B' helix, is achieved by an expansion of this region and a small displacement of the backbone. Interestingly, during the egress of testosterone along channel 1, Phe297 and Phe108 appear to act as two clamps to stabilize testosterone binding and prevent it from leaving the active site. Phe115 acts as a gatekeeper for channel 2. These results are in agreement with previous site-directed mutagenesis experiments.

Amino Acid Sequence↗

PubMed2005

Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide.

Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.

Amino Acid Substitution↗

PubMed2005

Functional role of residues in the helix B' region of cytochrome P450 2B1.

Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B' when ligands bind. To investigate the functional role of helix B' in P450 2B1, residues 100-109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresorufin, 7-ethoxy-4-trifluoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in k(cat) values and significant differences in catalytic efficiencies among the substrates examined, consistent with structural information suggesting that the helix B' region can adopt multiple conformations with different contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The findings suggest that residues in the helix B' region affect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry.

Amino Acid Substitution↗

PubMed2005

Structures of cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) catalyzes the initial step in the clearance of many pharmaceuticals and foreign chemicals. The structurally diverse nature of CYP3A4 substrates complicates rational prediction of their metabolism and identification of potential drug interactions. The first molecular structures of human CYP3A4 were recently determined, revealing an active site of sufficient size and topography to accommodate either large ligands or multiple smaller ligands, as suggested by the heterotropic and homotropic cooperativity of the enzyme.

Animals↗