Characterization of heme environment and mechanism of peroxide bond cleavage in human prostacyclin synthase

Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.     

Crystal structure of the human prostacyclin synthase

Prostacyclin synthase (PGIS) catalyzes an isomerization of prostaglandin H(2) to prostacyclin, a potent mediator of vasodilation and anti-platelet aggregation. Here, we report the crystal structure of human PGIS at 2.15 A resolution, which represents the first three-dimensional structure of a class III cytochrome P450. While notable sequence divergence has been recognized between PGIS and other P450s, PGIS exhibits the typical triangular prism-shaped P450 fold with only moderate structural differences. The conserved acid-alcohol pair in the I helix of P450s is replaced by residues G286 and N287 in PGIS, but the distinctive disruption of the I helix and the presence of a nearby water channel remain conserved. The side-chain of N287 appears to be positioned to facilitate the endoperoxide bond cleavage, suggesting a functional conservation of this residue in O-O bond cleavage. A combination of bent I helix and tilted B' helix creates a channel extending from the heme distal pocket, which seemingly allows binding of various ligands; however, residue W282, placed in this channel at a distance of 8.4 A from the iron with its indole side-chain lying parallel with the porphyrin plane, may serve as a threshold to exclude most ligands from binding. Additionally, a long "meander" region protruding from the protein surface may impede electron transfer. Although the primary sequence of the PGIS cysteine ligand loop diverges significantly from the consensus, conserved tertiary structure and hydrogen bonding pattern are observed for this region. The substrate-binding model was constructed and the structural basis for prostacyclin biosynthesis is discussed.     

Characterization of the substrate mimic bound to engineered prostacyclin synthase in solution using high-resolution NMR spectroscopy and mutagenesis: implication of the molecular mechanism in biosynthesis of prostacyclin

High-resolution NMR spectroscopy was used to determine the docking of a substrate (prostaglandin H2) mimic (U46619) to the engineered prostacyclin (PGI2) synthase (PGIS) in solution. The binding of U46619 to the PGIS protein was demonstrated by 1D NMR titration, and the significant perturbation of the chemical shifts of protons at C-11, H2C, and H20 of U46619 were observed upon U46619 binding to the engineered PGIS in a concentration-dependent manner. The detailed conformational change and 3D structure of the PGIS-bound U46619 were further demonstrated by 2D 1H NMR experiments using the transferred NOE technique. The distances between the protons H20 and H2, H18 and H2, and H18 and H4 are shorter following their binding to the PGIS in solution-down to within 5 A. These shorter distances resulted in a widely open conformation, where the triangle shape of the unbound U46619 changed to a more compact conformation with an oval shape. The bound conformation of U46619 fits the crystal structure of the PGIS substrate binding pocket considerably better than that of the unbound U46619. The residues important to the substrate binding in the active site pocket of PGIS were also predicted. For example, Trp282 could be one of the most important residues and is suspected to play a role in the determination of specific catalytic function, which has been established by the docking studies using the NMR structure of the PGIS-bound form of U46619 and the PGIS crystal structure. These studies have provided the structural information for the interaction of the PGIS with its substrate mimic. The noted conformational changes where the C-6 position is closer to the C-9 position of U46619 provided the first experimental data for understanding the molecular mechanism of the catalytic function of PGIS in the isomerization of PGH2 to prostacyclin.     

Purification and characterization of recombinant human prostacyclin synthase

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.     

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Cellular and molecular biology of prostacyclin synthase

Prostacyclin synthase (PGIS) cDNA comprises 1500 nucleotides coding for a 500 amino acid protein. It is a heme protein with spectral characteristics of cytochrome p450 (CYP). It does not possess the typical CYP mono-oxygenase activity but catalyzes the rearrangement of prostaglandin H2 to form PGI2. Analysis of its structure-function by molecular modeling and site-directed mutagenesis reveals a long substrate channel lined by hydrophobic residues. Cys-441 has been identified as the proximal axial ligand of heme. Tyr-430 is nitrated by peroxynitrite which results in reduced PGIS catalytic activity, suggesting that Tyr-430 is located close to the heme pocket. PGIS is constitutively expressed and may be upregulated by cytokines, reproductive hormones, and growth factors. It is physically colocalized with cyclooxygenases and phospholipases, and functionally coupled with these enzymes. PGIS coupling with COX-2 has been shown to play an important role in vascular protection, embryo development and implantation, and cancer growth.     

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.     

Identification of thromboxane synthase amino acid residues involved in heme-propionate binding

Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.     

Topology of catalytic portion of prostaglandin I(2) synthase: identification by molecular modeling-guided site-specific antibodies

Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.     

Conformational relaxation in hemoproteins: the cytochrome P-450cam case

Photodissociation of (CO)P-450(cam)(substrate) complexes was found to trigger a conformational relaxation process that interferes with ligand rebinding at temperatures as low as 140 K even though the protein conformational substates (CS(1)) remain frozen. To analyze the rebinding and relaxation kinetics, we developed a model that takes the distribution of relaxation rates explicitly into account and in which rebinding and relaxation rates are connected by a linear free energy relation. In all complexes heme relaxation occurs first and is probably faster than 100 ns even at 77 K. This is the only process found in substrate-free P-450(cam). Above 140 K and in the presence of a substrate, this initial, fast rebinding state (P) progressively relaxes to another state (P degrees ) in which rebinding is slower. The relaxation rate is independent of solvent rigidity and is governed by the protein's internal dynamics. Rebinding enthalpies in P and P degrees as well as the enthalpy shift brought about by relaxation correlate with the substrate propensity to block access to the iron site. In P degrees the barrier is higher because the substrate is closer to the heme normal and exerts more steric repulsion for CO binding. The relaxation process implies the return of substrate and heme to their ligand-free positions in which access to the heme is reduced.     

Crystal structure and properties of CYP231A2 from the thermoacidophilic archaeon Picrophilus torridus

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.     

Structural evidence for direct hydride transfer from NADH to cytochrome P450nor

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover.     

Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.     

Effects of ligand binding on dynamics of fatty acid binding protein and interactions with membranes

Intracellular transport of fatty acids involves binding of ligands to their carrier fatty acid binding proteins (FABPs) and interactions of ligand-free and -bound FABPs with membranes. Previous studies focused on ligand-free FABPs. Here, our amide hydrogen exchange data showed that oleic acid binding to human intestinal FABP (hIFABP) stabilizes the protein, most likely through enhancing the hydrogen-bonding network, and induces rearrangement of sidechains even far away from the ligand binding site. Using NMR relaxation techniques, we found that the ligand binding affects not only conformational exchanges between major and minor states but also the affinity of hIFABP to nanodiscs. Analyses of the relaxation and amide exchange data suggested that two minor native-like states existing in both ligand-free and -bound hIFABPs originate from global “breathing” motions, while one minor native-like state comes from local motions. The amide hydrogen exchange data also indicated that helix αII undergoes local unfolding through which ligands can exit from the binding cavity.     

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.     

The N-terminal membrane anchor domain of the membrane-bound prostacyclin synthase involved in the substrate presentation of the coupling reaction with cyclooxygenase

To mimic the native conditions, the cyclooxygenase (COX)/prostaglandin I(2) synthase (PGIS) coupling reaction system was used to determine the coordination of PGIS with COX for the biosynthesis of prostacyclin (PGI(2)) using arachidonic acid (AA) as a substrate in a membrane-bound environment. The membrane-bound PGIS exhibited a faster isomerization of PGH(2) produced by COX to PGI(2) than the detergent-solubilized PGIS. To determine whether the N-terminal domain of PGIS responds to the facilitation of PGH(2) movement (presentation) from COX to the active site of PGIS, the first 20 residues of PGIS (Delta20-PGIS) were deleted and expressed in COS-7 cells. Delta20-PGIS retained membrane-bound properties and exhibited a slower substrate presentation property. Furthermore, a chimeric molecule (PGIS/TXAS(8-27)) with the replacement of the first 20 residues of PGIS by the corresponding membrane anchor region (residues 8-27) of thromboxane A(2) synthase was created to evaluate the mechanism influencing the biosynthesis of PGI(2) in coordination with COX. The chimera revealed a multiple fold delay in the PGH(2) presentation in low range concentrations of AA (0.3-3muM) at 30s reactions. However, the delay could be recovered by a longer incubation time in high range concentrations of AA (>10muM), but not in low range concentrations of AA. These results demonstrated that the N-terminal domain of PGIS plays a role in the facilitation of the substrate presentation to the PGIS active site in low concentrations of AA, which may be a physiological condition. The TXAS N-terminal domain could not replace the function of the corresponding domain of PGIS, indicating that the facilitation of the substrate presentation is specific.     

Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form

Thromboxane A2 synthase (TXAS) binds to the endoplasmic reticulum membrane and catalyzes both an isomerization of prostaglandin H2 (PGH2) to form thromboxane A2 (TXA2) and a fragmentation of PGH2 to form 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA). TXAS is a non-classic cytochrome P450 in that it does not require molecular oxygen or an external electron donor for catalysis. Difficulty in obtaining crystals from the membrane-bound TXAS prompted us to modify the protein to a soluble form. Results from site-directed mutagenesis, hydropathy analysis, and homology modeling led us to identify a putative membrane association segment near the end of helix F in TXAS. We report here the generation of a soluble form of TXAS by deletion of the amino-terminal membrane-anchoring domain and replacement of the helix F and F-G loop region with the corresponding region of the structurally characterized microsomal P450 2C5. The resultant TXAS/2C5 chimera is expressed in bacteria as a cytosolic and monomeric protein. Addition of an amino-terminal leader sequence to enhance expression and a tetra-histidine segment at the carboxyl-terminus to facilitate purification yielded approximately 4 mg of nearly homogeneous TXAS/2C5 per liter of bacterial culture. The TXAS/2C5 chimera contains heme at nearly a 1:1 molar ratio and catalyzes the formation of TXA2, MDA, and HHT at a 1:1:1 ratio, although with a reduced catalytic activity compared to wild type TXAS. TXAS/2C5 exhibits electronic absorption spectra similar to wild type TXAS and has similar affinities toward distal heme ligands such as imidazole and U44069. The chimera was mono-dispersive and thus is promising for crystallization trials.     

Two-dimensional NMR and All-atom Molecular Dynamics of Cytochrome P450 CYP119 Reveal Hidden Conformational Substates

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [¹³C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.     

Cytochrome P450 from Photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site

We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further toward the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoylglycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoylglycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around -100 to -200 mL/mol and P(1/2) of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium toward the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.