Studies on Regioselective Binding Mode of Steroid Molecules in Homology Modeled Cytochrome P450-2C11

In this study, we investigated the regioselective binding mode of steroid molecules and structure requirements for steroid molecules for 16[alpha]-hydroxylation by Cytochrome P450-2C11. Docking study by using the homology Cytochrome P450-2C11 indicated that 16[alpha]-hydroxylation is favored with steroidal molecules possessing the following components, 1) a bent A-B ring configuration (5[beta]-reduced), 2) C-3[alpha]-hydroxyl group, 3) C-17[beta]-acetyl group, and 4) methyl group at both the C-18 and C-19. These respective steroid components requirements such as A-B ring configuration and functional groups at C-3 and C-17 were defined as the inhibitory contribution factor. Overall results by rat CYP2C11 revealed that steroidal structure requirements resulted in causing an effective inhibition of [^3^H]progesterone 16[alpha]-hydroxylation by the adult male rat liver microsome. As far as docking of homology modeled CYP2C11 against investigated steroids is concerned, they are docked at the active site superimposed with flurbiprofen. It was also found that the distance between heme iron and C16[alpha]-H was between 4 to 6 Å and that the related angle was in the range of 180±45°

Enzymatic functionalization of carbon-hydrogen bonds

The development of new catalytic methods to functionalize carbon–hydrogen (C–H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C–H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C–H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C–H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references)

Catalysts on Demand: Selective Oxidations by Laboratory-Evolved Cytochrome P450 BM3

Efficient catalysts for selective oxidation of C-H bonds using atmospheric oxygen are highly desirable to decrease the economic and environmental costs associated with conventional oxidation processes. We have used methods of directed evolution to generate variants of bacterial cytochrome P450 BM3 that catalyze hydroxylation and epoxidation of a wide range of nonnative substrates. This fatty acid hydroxylase was converted to a propane monooxygenase (PMO) capable of hydroxylating propane at rates comparable to that of BM3 on its natural substrates. Variants along the PMO evolutionary lineage showed broadened substrate scope; these became the starting points for evolution of a wide array of enzymes that can hydroxylate and derivatize organic scaffolds. This work demonstrates how a single member of enzyme family is readily converted by evolution into a whole family of catalysts for organic synthesis

Hydroxylation Structure and Proton Transfer Reactivity at the Zinc Oxide-Water Interface

The hydroxylation structural features of the first adsorption layer and its connection to proton transfer reactivity have been studied for the ZnO-liquid water interface at room temperature. Molecular dynamics simulations employing the ReaxFF forcefield were performed for water on seven ZnO surfaces with varying step concentrations. At higher water coverage a higher level of hydroxylation was found, in agreement with previous experimental results. We have also calculated the free energy barrier for transferring a proton to the surface, showing that stepped surfaces stabilize the hydroxylated state and decrease the water dissociation barrier. On highly stepped surfaces the barrier is only 2 kJ/mol or smaller. Outside the first adsorption layer no dissociation events were found during almost 100 ns of simulation time; this indicates that these reactions are much more likely if catalyzed by the metal oxide surface. Also, when exposed to a vacuum, the less stepped surfaces stabilize adsorption beyond monolayer coverage

Life fingerprints of nuclear reactions in the body of animals

Nuclear reactions are a very important natural phenomenon in the universe. On the earth, cosmic rays constantly cause nuclear reactions. High energy beams created by medical devices also induce nuclear reactions in the human body. The biological role of these nuclear reactions is unknown. Here we show that the in vivo biological systems are exquisite and sophisticated by nature in influence on nuclear reactions and in resistance to radical damage in the body of live animals. In this study, photonuclear reactions in the body of live or dead animals were induced with 50-MeV irradiation. Tissue nuclear reactions were detected by positron emission tomography (PET) imaging of the induced beta+ activity. We found the unique tissue "fingerprints" of beta+ (the tremendous difference in beta+ activities and tissue distribution patterns among the individuals) are imprinted in all live animals. Within any individual, the tissue "fingerprints" of 15O and 11C are also very different. When the animal dies, the tissue "fingerprints" are lost. The biochemical, rather than physical, mechanisms could play a critical role in the phenomenon of tissue "fingerprints". Radiolytic radical attack caused millions-fold increases in 15O and 11C activities via different biochemical mechanisms, i.e. radical-mediated hydroxylation and peroxidation respectively, and more importantly the bio-molecular functions (such as the chemical reactivity and the solvent accessibility to radicals). In practice biologically for example, radical attack can therefore be imaged in vivo in live animals and humans using PET for life science research, disease prevention, and personalized radiation therapy based on an individual's bio-molecular response to ionizing radiation

A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

In vitro metabolic studies of REV-ERB agonists SR9009 and SR9011

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC-HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended

A fundamental mechanism for carbon-film lubricity identified by means of ab initio molecular dynamics

Different hypotheses have been proposed to explain the mechanism for the extremely low friction coefficient of carbon coatings and its undesired dependence on air humidity. A decisive atomistic insight is still lacking because of the difficulties in monitoring what actually happens at the buried sliding interface. Here we perform large-scale ab initio molecular dynamics simulations of both undoped and silicon-doped carbon films sliding in the presence of water. We observe the tribologically-induced surface hydroxylation and subsequent formation of a thin film of water molecules bound to the OH-terminated surface by hydrogen bonds. The comparative analysis of silicon-incorporating and clean surfaces, suggests that this two-step process can be the key phenomenon to provide high slipperiness to the carbon coatings. The water layer is, in fact, expected to shelter the carbon surface from direct solid-on-solid contact and make any counter surface slide extremely easily on it. The present insight into the wettability of carbon-based films can be useful for designing new coatings for biomedical and energy-saving applications with environmental adaptability.Comment: 22 pages, 4 figures, 1 tabl