Crystal structure of CYP199A2, a para-substituted benzoic acid oxidizing cytochrome P450 from Rhodopseudomonas palustris

CYP199A2, a cytochrome P450 enzyme from Rhodopseudomonas palustris, oxidatively demethylates 4-methoxybenzoic acid to 4-hydroxybenzoic acid. 4-Ethylbenzoic acid is converted to a mixture of predominantly 4-(1-hydroxyethyl)-benzoic acid and 4-vinylbenzoic acid, the latter being a rare example of CC bond dehydrogenation of an unbranched alkyl group. The crystal structure of CYP199A2 has been determined at 2.0-Å resolution. The enzyme has the common P450 fold, but the B′ helix is missing and the G helix is broken into two (G and G′) by a kink at Pro204. Helices G and G′ are bent back from the extended BC loop and the I helix to open up a clearly defined substrate access channel. Channel openings in this region of the P450 fold are rare in bacterial P450 enzymes but more common in eukaryotic P450 enzymes. The channel is hydrophobic except for the basic residue Arg246 at the entrance, which probably plays a role in the specificity of this enzyme for charged benzoates over neutral phenols and benzenes. The substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues. Computer docking of 4-ethylbenzoic acid into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the β-methyl group is located over the heme iron by Phe185, the side chain of which is only 6.35 Å above the iron in the native structure. This binding orientation is consistent with the observed product profile of exclusive attack at the para substituent. Putidaredoxin of the CYP101A1 system from Pseudomonas putida supports substrate oxidation by CYP199A2 at ∼6% of the activity of the physiological ferredoxin. Comparison of the heme proximal faces of CYP199A2 and CYP101A1 suggests that charge reversal surrounding the surface residue Leu369 in CYP199A2 may be a significant factor in this low cross-activity.     

Facile production of minor metabolites for drug development using a CYP3A shuffled library

Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11±4 (mean±SD) recombinations and 1±1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6 L batch culture of one such mutant enabled the facile isolation of 0.3 mg of the minor metabolite 1β-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.     

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for the first time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe120 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe120 in binding dextromethorphan and MDMA.     

Apo-cytochrome b5 as an indicator of changes in heme accessability: preliminary studies with cytochrome P450 3A4.

Cytochrome P450s (P450 or CYP) are the largest family of hemeproteins yet characterized. X-ray crystallographic studies have shown that the heme of the P450 hemeproteins is buried in the interior of the protein molecule. Unexplored are answers to questions concerning the role of heme in the folding of newly synthesized apo-P450s and the factors that influence changes in heme accessibility following modification of the pattern of folding of the holo-P450s. We have carried out the present studies to measure changes in heme accessibility in P450s. This is an initial step to determining whether heme-binding confers structural and functional integrity and stability to a P450 molecule. Recently, we have shown that apo-high molecular weight cytochrome b5 (apo-HMWb5) is an efficient acceptor of heme when added to a preparation of purified recombinant CYP3A4. In the present work we have studied heme binding by apo-HMWb5 when mixed with a number of different hemeproteins (myoglobin, hemoglobin, catalase, CYP4A1, CYP101, and CYP3A4). These hemeproteins differ in the location of the heme (i.e., surface or internal) allowing one to study changes in structure as measured by the process of heme transfer from one protein to another. It was found that heme transfer to apo-HMWb5 occurs relatively rapidly from hemeproteins where the heme is located at or near the surface or when the hemeprotein is denatured. In contrast, heme transfer from P450s to apo-HMWb5 occurs only following modification of the P450 structure with chaotropic agents. An exception is CYP3A4 where a measurable amount of heme is transferred to apo-HMWb5 in the absence of denaturing agents. The preliminary results described here employs apo-HMWb5 as an indicator for assessing changes in heme-availability of P450s as the protein-folding of the molecule is altered.     

Enzymatic properties of cytochrome P450 catalyzing 3′-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3 kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3′-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Molecular modeling and identification of substrate binding site of orphan human cytochrome P450 4F22

Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human cytochrome P450 4F22 (CYP4F22) was categorized into "orphan" CYPs because of its unknown function. CYP4F22 is a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22 remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the biological roles of CYP4F22 and structure-based drug design.     

Crystal Structure of CYP24A1, a Mitochondrial Cytochrome P450 Involved in Vitamin D Metabolism

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 Å resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A′ and G′, suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1α,25-dihydroxyvitamin D₃ binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K″, and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.     

Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: the molecular basis of substrate binding and regioselectivity toward warfarin

To examine the molecular basis of activity and regioselectivity of the clinically important human microsomal cytochrome P450 (CYP) monooxygenase 2C9 toward its substrate warfarin, 22 molecular dynamics simulations (3-5 ns each) were performed in the presence and absence of warfarin. The resulting trajectories revealed a stable protein core and mobile surface elements. This mobility leads to the formation of two surface channels in the region between F-G loop, B' helix/B-B' loop, beta(1)-sheet, and between helices F and I and the turn in the C-terminal antiparallel beta-sheet in the presence of warfarin. Besides the nonproductive state of the CYP2C9 warfarin complex captured in the crystal structure, three additional states were observed. These states differ in the shape of the substrate binding cavity and the position of the warfarin molecule relative to heme. In one of these states, the 7- and 6-positions of warfarin contact the heme with a marked geometrical preference for position 7 over position 6. This modeling result is consistent with experimentally determined regioselectivity (71 and 22% hydroxylation in positions 7 and 6, respectively). Access to the heme group is limited by the core amino acids Ala297, Leu362, Leu366, and Thr301, which therefore are expected to have a major impact on regioselectivity. In addition, modeling predicts that autoactivation of warfarin is sterically hindered. Our study demonstrates how the combination of mobile surface and rigid core leads to interesting properties: a broad substrate profile and simultaneously a high regioselectivity.     

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.     

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.     

Crystal structure of the Mycobacterium tuberculosis P450 CYP121-fluconazole complex reveals new azole drug-P450 binding mode

Azole and triazole drugs are cytochrome P450 inhibitors widely used as fungal antibiotics and possessing potent antimycobacterial activity. We present here the crystal structure of Mycobacterium tuberculosis cytochrome P450 CYP121 in complex with the triazole drug fluconazole, revealing a new azole heme ligation mode. In contrast to other structurally characterized cytochrome P450 azole complexes, where the azole nitrogen directly coordinates the heme iron, in CYP121 fluconazole does not displace the aqua sixth heme ligand but occupies a position that allows formation of a direct hydrogen bond to the aqua sixth heme ligand. Direct ligation of fluconazole to the heme iron is observed in a minority of CYP121 molecules, albeit with severe deviations from ideal geometry due to close contacts with active site residues. Analysis of both ligand-on and -off structures reveals the relative position of active site residues derived from the I-helix is a key determinant in the relative ratio of on and off states. Regardless, both ligand-bound states lead to P450 inactivation by active site occlusion. This previously unrecognized means of P450 inactivation is consistent with spectroscopic analyses in both solution and in the crystalline form and raises important questions relating to interaction of azoles with both pathogen and human P450s.     

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 A reveals novel features of cytochrome P450

The first structure of a P450 to an atomic resolution of 1.06 A has been solved for CYP121 from Mycobacterium tuberculosis. A comparison with P450 EryF (CYP107A1) reveals a remarkable overall similarity in fold with major differences residing in active site structural elements. The high resolution obtained allows visualization of several unusual aspects. The heme cofactor is bound in two distinct conformations while being notably kinked in one pyrrole group due to close interaction with the proline residue (Pro(346)) immediately following the heme iron-ligating cysteine (Cys(345)). The active site is remarkably rigid in comparison with the remainder of the structure, notwithstanding the large cavity volume of 1350 A(3). The region immediately surrounding the distal water ligand is remarkable in several aspects. Unlike other bacterial P450s, the I helix shows no deformation, similar to mammalian CYP2C5. In addition, the positively charged Arg(386) is located immediately above the heme plane, dominating the local structure. Putative proton relay pathways from protein surface to heme (converging at Ser(279)) are identified. Most interestingly, the electron density indicates weak binding of a dioxygen molecule to the P450. This structure provides a basis for rational design of putative antimycobacterial agents.     

Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.     

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily ω-1 and ω-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.     

Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at "hot spots" of protein-protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.     

FTIR studies of the redox partner interaction in cytochrome P450: The Pdx–P450cam couple

Recently we have developed a new approach to study protein–protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam–Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP–FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in β-sheets and α-helix content, a decrease in the population of random coil/3₁₀-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam–Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx–P450cam complex.