Cytochrome P450 1A-ligand interactions: Implications for substrate specificity and inhibitor susceptibility

Cytochromes P450 are heme-containing enzymes that are involved in the metabolism of a variety of clinically important drugs, endogenous and exogenous compounds, including a number of procarcinogens. P450 1A subfamily has two members: 1A1 and 1A2. P450 1A1 and 1A2 show high sequence identity (\u3e70%), but display different substrate specificity and inhibitor susceptibility. P450 1A2 is one of the major hepatic P450s, which metabolizes more than 11% of drugs currently on the market. Thus, we focused our attention on studies of this particular P450.;The five key active site residues that are different between P450 1A1 and 1A2 have been proposed to play an important role in determining the substrate binding orientation. We adopted phenacetin, an important substrate marker for P450 1A2, to investigate this role. Kinetic studies have shown that the L382V mutant and other mutants containing the L382V substitution exhibited markedly higher catalytic efficiency than the wild type enzyme, while other four single mutants displayed much lower activity. Stoichiometry studies indicated that the higher coupling occurred due to decreased water formation in the catalytic cycle by L382V and mutants containing the L382V substitution. Docking and molecular dynamic simulations suggested that the L382V substitution enabled the oxidation site of phenacetin to move closer to the ferryl oxygen of heme, thereby promoting phenacetin metabolism.;In order to verify the above mechanism, NMR T1 relaxation measurements were utilized to estimate the distance between protons of phenacetin and ferryl oxygen of oxo-heme of P450 wild type or mutants. The results showed that the time-averaged orientations of phenacetin in the active site were very similar in P450 1A2 wild type and mutants. However, the protons at the site of oxidation of phenacetin were closer to the ferryl oxygen in P450 1A2 L382V and L382V/N312L mutants than P450 1A2 WT, which is consistent with the findings from molecular modeling.;To extend our studies, we explored the interactions between inhibitors and P450 1A2 WT and mutants. Molecular modeling techniques, including docking and molecular dynamic simulations, have been extensively used to predict possible inhibitor-enzyme interactions and describe the docking energy involved. In some cases, for example with residue Phe226, pi-pi stacking might play a major role in these interactions. Good correlations between docking scores and inhibition constants Ki were obtained using AutoDock program.;The combination of molecular modeling and experimental techniques helped us to thoroughly investigate the structure-function relationships of P450 1A2. The insight we gained into the catalytic and inhibition mechanism(s) of this enzyme stresses the importance of the active site topology for P450 activity and provides important implications for the rational design of anticancer drugs

Significant increase in phenacetin oxidation on L382V substitution

ABSTRACT: Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/ T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/ N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wildtype enzyme, with k cat values 3-fold higher, in contrast to other mutants, for which k cat decreased. Likewise, a significant increase in specificity, expressed as the k cat /K m ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity. Cytochromes P450 (P450s) are heme-containing monooxygenase enzymes, which are involved in the metabolism of numerous exogenous and endogenous compounds. P450s are ubiquitous in living organisms, with at least 50 families and 82 subfamilies found in different species. Human CYP1A subfamily has two major isoforms: CYP1A1 and CYP1A2. CYP1A2, one of the major P450s in the human liver, was first characterized as a phenacetin O-deethylase In humans, CYP1A2 shares 72% amino acid sequence identity with CYP1A1, but the substrate specificities and inhibitor susceptibilities of these enzymes are different. For example, substrates such as phenacetin and 7-methoxyresorufin are primarily metabolized by CYP1A2 with high catalytic efficiency, whereas CYP1A1 displays weak capability to oxidize those substrates. On the other hand, 7-ethoxyresorufin is preferentially oxidized by CYP1A1 Our previous studies on structure-function relationships of CYP1A1 indicated that Val382 played an important role in binding of alkoxyresorufin substrates In the current study, we chose phenacetin as a substrate. This compound has been used as the most common marker for CYP1A

Role of Mineral Nutrients in Plant-Mediated Synthesis of Three-Dimensional Porous LaCoO3

With the assistance of plant extracts, the facile synthesis of three-dimensional (3D) porous LaCoO3 perovskite is reported at a lower calcination temperature of 500 °C. The formation mechanism is carefully studied by investigating the different roles of organic and inorganic components in Cacumenplatycladi extract. The results indicate that organic components (mainly phenolic acids) function as the similar complex species of citric acid, while the mineral nutrients (Na+, K+, Ca2+, and Mg2+) together with NO3– serve as combustion-aid agents even with trace amounts. Moreover, the biosynthesized LaCoO3 has a high surface area of 32.5 m2 g–1 and exhibits excellent catalytic performance for benzene oxidation. Benzene of 1000 ppm can achieve a stable conversion above 90% at 285 °C in a continuous run for 80 h (weight-hourly space velocity (WHSV) = 40 000 mL g–1 h–1). It can be attributed to the bio-LaCoO3 with more electrophilic adsorption of oxygen species and 3D porous structure

Effects of N-Terminal Non-catalytic Domains on Enzymatic Properties of the Alginate Lyase AlgL7 from Microbulbifer sp. ALW1

In order to clarify the effect of the non-catalytic carbohydrate-binding module (CBM) and F5/8 type C domains on the enzymatic properties of AlgL7, an alginate lyase from Microbulbifer sp. ALW1, the full-length enzyme AlgL7 and two truncated enzymes: CD1 (catalytic domain) and CD2 (containing F5/8 type C domain and catalytic domain) were constructed and characterized. The results showed that the truncated enzyme CD2 exhibited higher specific activity, thermostability, Michaelis constant (Km), and maximum reaction velocity (Vmax) compared to the full-length AlgL7, indicating that the CBM domain played an important role in maintaining the substrate affinity of the enzyme, but reduced the catalytic activity, thermostability, and Vmax value the enzyme. Compared to the truncated enzyme CD1, CD2 exhibited higher specific activity, optimal reaction temperature, thermostability, Vmax, and Km, indicating that the F5/8 type C domain contributed to improve the enzymatic activity, optimum reaction temperature, thermostability, and Vmax, but reduced the substrate affinity of the enzyme. Using sodium alginate as the substrate, the specific activity of CD2 was 183.9 U/mg. The optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The Km and Vmax were 39.80 mg/mL and 2 000 U/mg, respectively. The major enzymatic hydrolysates were disaccharides and trisaccharides. This study promotes the understanding of the structure-activity relationship between the non-catalytic domains and the properties of alginate lyase, and lays a theoretical basis for using the non-catalytic domains to improve the catalytic properties of alginate lysate

Bioelectricity generation from the decolorization of reactive blue 19 by using microbial fuel cell.

Microbial fuel cell (MFC) was compared to conventional biological techniques for decolorization of anthraquinone dye, reactive blue 19 (RB19) with simultaneous electricity generation. With 50 mg/L of RB19 in the anode chamber as a fuel, the MFC achieved 89% decolorization efficiency of RB19 within 48 h, compared with 51 and 55% decolorization efficiency achieved by aerobic and anaerobic techniques, respectively. The cyclic voltammetry results showed that RB19 could promote the electron transfer and redox reaction on the surface of anode. The RB19 decolorization process can be described by first-order kinetics, and the decolorization rate decreased with the increase of RB19 concentration. The high-throughput 16S rRNA sequencing analysis indicated significant microbial community shift in the MFC. At phylum level, the majority of sequences belong to Proteobacteria, accounting from 23 to 84% of the total reads in each bacterium community. At genus level, the MFC contained two types of microorganisms in general such as electrochemically active and decolorization bacteria. Overall, MFC is an effective method for anthraquinone dye treatment with simultaneous energy recovery. The 16S rRNA revealed that there were two major functioning microbial communities in the MFC such as electricity-producing and RB19-degrading bacteria which synergistically worked on RB19 degradation

Green synthesis of Au-Pd bimetallic nanoparticles: Single-step bioreduction method with plant extract

A facile and eco-friendly method for the preparation of Au-Pd bimetallic nanoparticles (similar to 7 nm) has been developed based on simultaneous bioreduction of Au(III) and Pd(II) precursors with Cacumen Platycladi leaf extract in aqueous environment. The morphology, structure, and size were confirmed with the aid of transmission electron microscopy, selected area electron diffraction. UV-vis spectroscopy, X-ray diffraction, and energy dispersive X-ray spectroscopy. The results from Fourier transform infrared spectroscopy showed that the C=O and C-O groups in the plant extract played a critical role in capping the nanoparticles. Importantly, the process can be described as pure "green chemistry" technique since no additional synthetic reagents are used as reductants or stabilizers. (C) 2011 Elsevier B.V. All rights reserved.National Natural Science Foundation of China[21036004, 20976146]; Natural Science Foundation of Fujian Province of China[2010J05032, 2010J01052]WOS:00029506830004

Microwave-Assisted Biosynthesis of Ag/ZrO2 Catalyst with Excellent Activity toward Selective Oxidation of 1,2-Propanediol

通讯作者地址: Huang, JLIn the biorefining process, polyols are important intermediates, and the oxidation of polyols toward other value added products is of great significance. This work describes a green and facile biosynthesis method for the preparation of Ag/ZrO2 catalyst for selective oxidation of 1,2-propanediol (a typical polyol). Cinnamomum comphora (CC) leaf extract was employed as the reducing and capping agent for the preparation of Ag nanoparticles (NPs) with the assistance of microwave irradiation. The main reducing agents were identified as polyphenols by Fourier transform infrared spectroscopic analysis of CC extracts before and after reaction. After electrostatic adsorption, the NPs were anchored onto the support ZrO2. The Ag/ZrO2 catalysts were found with good dispersity and showed excellent activity toward selective oxidation of 1,2-propanediol. The effects of the preparation conditions on catalyst activity were studied; the optimal condition was obtained (microwave time of 4 min, CC concentration of 12 g/L and Ag loading of 5%). Since the natural capping agents are easy to remove, the catalysts need no calcination treatment before catalytic reaction. Thus, the microwave-assisted biosynthesis appears to be environmentally benign as neither expensive chemicals nor intensive energy consumption is engaged.National Nature Science Foundation 21036004 2120614

Plant-mediated fabrication and surface enhanced raman property of flower-like Au@Pd nanoparticles

The flower-like nanostructures of an Au core and Pd petals with the average size of 47.8 nm were fabricated through the successive reduction of HAuCl4 and Na2PdCl4 at room temperature. During the synthesis, Cacumen Platycladi leaf extract served as weak reductant and capping agent. Characterization techniques such as Energy-dispersive X-ray spectroscopy, UV-Vis spectroscopy, and X-ray diffraction characterizations were employed to confirm that the as-synthesized nanoparticles have the structure of core-shell. The obtained core-shell nanoflowers exhibited good surface enhanced Raman spectroscopic activity with Rhodamine 6G. ? 2014 by the authors