Resolution of rabbit liver cytochrome P450 3b and 3c (P450 IIC3 and IIIA4) from microsomal membrane by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was applied to the separation of microsomal proteins and P450 isozymes. The experimental conditions of the first-dimensional isoelectric focusing were optimized. The best results were obtained under the following conditions: (i) a mixture of two detergents (Tergitol NP 10 and CHAPS) in the focusing gel and sample buffer, (ii) a carrier ampholyte gradient between pH 6.5 and 9.0, (iii) sample loading at the anodic end of the gel, and (iv) low protein loading in the sample (below 50 micrograms). Four P450 forms, including P450 3b and 3c, could be resolved under these conditions.     

Structure-based design,synthesis of novel probes for cytochrome P450 OleT

Cytochrome P450 OleT_SA, a new cytochrome P450 enzyme from Staphylococcus aureus, catalyzes the oxidative decarboxylation and hydroxylation of fatty acids to generate terminal alkenes and fatty alcohols. The mechanism of this bifurcative chemistry remains largely unknown. Herein, a class of derivatized fatty acids were synthesized as probes to investigate the effects of substrate structure on the product type of P450 OleT_SA. The results demonstrate that the fine-tuned structure of substrates, even in a remote distance from the carboxyl group, significantly regulates OleT catalyzed decarboxylation/hydroxylation reactions. Molecular docking analysis indicated the potential interactions between the carboxylate groups of different probes and the enzyme active center which was attributed to the bifurcative chemistry.     

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.     

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide prodrug

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an overall yield of 26% in four steps from prolinol is reported; the previously published yield of this transformation was 1%. Using this revised strategy, the sarcosine analogue (14) of V-PROLI/NO was prepared. Finally, the methyl ester of V-PROLI/NO (15) was found to be an esterase-activated prodrug form of V-PROLI/NO.     

Taxol biosynthesis: Molecular cloning and characterization of a cytochrome P450 taxoid 7 beta-hydroxylase

Biosynthesis of the anticancer drug Taxol in yew species involves eight cytochrome P450-mediated oxygenations and four coenzyme A-dependent acylations of the diterpenoid core. A family of cytochrome P450 genes, obtained from a yew cell cDNA library, were functionally expressed and screened with taxusin (taxa-4(20),11(12)-dien-5 alpha,9 alpha,10 beta,13 alpha-tetraol tetraacetate) as a surrogate substrate. One clone converted this substrate to an oxygenated derivative that was identified as 7 beta-hydroxytaxusin. The structure and properties of this 7 beta-hydroxylase are similar to those of other taxoid hydroxylases. Kinetic and binding assays indicated selectivity of the 7 beta-hydroxylase for polyoxygenated and acylated taxoid substrates, an observation consistent with the operation of this enzyme in the central portion of the Taxol biosynthetic pathway. Although the 7 beta-hydroxyl of Taxol is not essential for antimitotic activity, this functional group provides a convenient means for preparing taxoid derivatives.     

Enantioselective epoxidation of linolenic acid catalysed by cytochrome P450(BM3) from Bacillus megaterium

Cytochrome P450(BM3), from Bacillus megaterium, catalyses the epoxidation of linolenic acid yielding 15,16-epoxyoctadeca-9,12-dienoic acid with complete regio- and moderate enantio-selectivity (60% ee). The absolute configuration of the product is tentatively assigned as 15(R),16(S)-. The Michaelis-Menten parameters kcat and Km for the reaction were determined to be 3126 +/- 226 min(-1) and 24 +/- 6 microM respectively.     

Oxidation of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient

Oxidations of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient were studied. The only product formed in Compound I oxidations was 10,11-epoxyundecanoic acid, whereas the enzyme under turnover conditions gave the epoxide and 9-hydroxy-10-undecenoic acid in a 10 : 90 ratio. Kinetic studies at 0 °C of oxidations by Compounds I formed by MCPBA oxidation and by a photo-oxidation pathway gave the same results, displaying saturation kinetics that yielded equilibrium binding constants and first-order oxidation rate constants that were experimentally indistinguishable. Oxidation of 10-undecenoic acid by Compound I from CYP119 generated by MCBPA oxidation also gave 10,11-epoxyundecanoic acid as the only product. CYP119 Compound I bound the substrate less strongly but reacted with a faster oxidation rate constant than P450(BM-3) Compound I. The kinetic parameters for oxidation of the substrate by P450(BM-3) under turnover conditions were similar to those of the Compound I transient even though the products differed.     

Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3

Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.     

High-throughput approaches to the quantitative analysis of ketoconazole, a potent inhibitor of cytochrome P450 3A4, in human plasma

Ketoconazole, an imidazole-piperazine compound, is an orally active antimycotic agent. In addition, ketoconazole is a specific inhibitor of cytochrome P450 3A4. As about 60% of oxidized drugs are biotransformed by this isoform, the potential effect of a concomitant administration of ketoconazole on drug disposition may be of interest during drug development. The present paper describes three different approaches (methods A, B, and C) to attain high-throughput sample preparation and analysis in the quantification of ketoconazole in human plasma. Method A consisted of acetonitrile precipitation in a 96-well plate, transfer of the supernatant via a Tomtec Quadra 96 Model 320, and subsequent injection onto a 50 x 4.6 mm (i.d.) Develosil Combi-RP-5 column (packed with C30 bonded silica particles). Method B consisted of an identical sample preparation to method A with the exception that a Michrom Magic Bullet(trade mark) column, 2.0 --> 0.50 mm (i.d., tapered bore) x25 mm length, was used. Lastly, in method C, a turbulent-flow chromatography (TurboFlow LC/APCI-MS/MS) module was used for the direct analysis of ketoconazole in human plasma. A Sciex API 3000 was used in methods A and B, while a Micromass Quattro LC was employed in method C. Based on the values obtained for the calibrator (standard) and quality control samples, all three protocols yielded satisfactory accuracy, precision, and reduced manual sample preparation time.     

Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH), however, their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells. A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system. Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH...     

MNDO-PM3 study on model cytochrome P450-mediated desulfuration of thiophosphoryl trifluoride,trimethylphosphine sulfide,and trimethyl phosphorothionate

The reaction mechanism of the model cytochrome P450-mediated desulfuration of thiophosphoryl trifluoride, trimethylphosphine sulfide, and trimethyl phosphorothionate was examined by the semiempirical MNDO-PM3 molecular orbital method. The proposed pathways, involving initial formation of S-oxide followed by rear-rangement to a cyclic phosphoxathiirane and the successive liberation of atomic sulfur to form a phosphate derivative, wee stepwisely analyzed by using the two model active oxygen species, O(¹D) and O(³P). Reaction coordinate analysis followed by refinement of transition states in each spin state strongly suggested that the desulfuration of thiophosphorus compounds by O(³P) was energetically more favorable than O(¹D). The alternative triplet pathway, abstraction of sulfur by O(³P) forming phosphine and sulfur monoxide followed by recombination between phosphorus and oxygen, was found to be energetically more probable than the rearrangement pathway. © 1993 by John Wiley & Sons, Inc.     

Reversible pressure deformation of a thermophilic cytochrome P450 enzyme (CYP119) and its active-site mutants

The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-site Thr213 and Thr214 mutants was investigated. At 20 degrees C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volume of activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolute reaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480 MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decomposition of iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variation of the N-arylprotoporphyrin-IX regioisomer (N(B):N(A):N(C):N(D)) adduct pattern from 39:47:07:07 at 0.1 MPa to 23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation and decomposition gave the same regioisomer distribution as untreated protein. The results indicate that the protein is reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450(cam) and other P450 enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.     

Syntheses,crystal structures,and cytochrome P450-like hydroxylation property of two novel copper(II) complexes

Two new copper(II) complexes, [Cu₂ (Phen)₂ (₂ -OH)₂ ((HCO)₃) ₂ ][Cu₂ (Phen) ₂ ( ₂ -OH)₂ (H₂ O)₂](dhba)₂ .8H₂ O (1) and [(bz)₄ Cu₃ (_2-OH)₂ (ppz)]_n (2) (where Phen = 1,10-phenanthroline, dhba = 2,6-dihydroxy-benzoate anion, bz = benzoate anion, and ppz = piperazine), have been synthesized and characterized by elemental analyses, i.r. and single crystal X-ray diffraction. Complex (1) is a tetra-nuclear Cu^II compound; each Cu^II atom in the complex has a square-pyramidal geometry, being coordinated by two nitrogen atoms and three oxygen atoms. Complex (2) is a trinuclear Cu^II compound. The Cu1 atom in the complex has a slightly distorted square plane, being coordinated by four oxygen atoms, while the Cu2 atom has a severely distorted square pyramid geometry, being coordinated by four oxygen atoms and one nitrogen atom. The determination studied by GC/MS analysis indicated that complex (1) has good catalytic activity for the dioxygen oxidization of cyclohexane.     

Design, synthesis and antitumor activity of a series of novel coumarin-stilbene hybrids, the 3-arylcoumarins

A series of novel coumarin-stilbene hybrids called 3-arylcoumarins were synthesized via Perkin reaction and evaluated as potential antitumor agents. The results showed that some compounds exhibited in vitro activity against KB, KV, MCF-7, MCF-7/ADR cell lines to some extent. Compound 3a showed remarkable effect against KB tumor cells with an IC50 value of 5.18 μmol/L.     

Design, synthesis and antitumor activity of a series of novel coumarin-stilbenes hybrids, the 3-arylcoumarins

<正>A series of novel coumarin-stilbenes hybrids called 3-arylcoumarins were synthesized via Perkin reaction and evaluated as potential antitumor agents.The results showed that some compounds exhibited in vitro activity against KB,KV,MCF-7,MCF-7/ ADR cell lines to some extent.Compound 3a showed remarkable effect against KB tumor cells with an IC_(50) value of 5.18μmol/L.