Investigating and Engineering Enzymes by Genetic Selection

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.     

Photobiocatalysis: Activating Redox Enzymes by Direct or Indirect Transfer of Photoinduced Electrons

Biocatalytic transformation has received increasing attention in the green synthesis of chemicals because of the diversity of enzymes, their high catalytic activities and specificities, and mild reaction conditions. The idea of solar energy utilization in chemical synthesis through the combination of photocatalysis and biocatalysis provides an opportunity to make the “green” process greener. Oxidoreductases catalyze redox transformation of substrates by exchanging electrons at the enzyme′s active site, often with the aid of electron mediator(s) as a counterpart. Recent progress indicates that photoinduced electron transfer using organic (or inorganic) photosensitizers can activate a wide spectrum of redox enzymes to catalyze fuel‐forming reactions (e.g., H₂ evolution, CO₂ reduction) and synthetically useful reductions (e.g., asymmetric reduction, oxygenation, hydroxylation, epoxidation, Baeyer–Villiger oxidation). This Review provides an overview of recent advances in light‐driven activation of redox enzymes through direct or indirect transfer of photoinduced electrons.     

Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC–MS/MS (ultrahigh-pressure liquid chromatography–tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t_1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The V_max of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the V_max of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication–drug and drug–enzyme interactions.     

Engineering of Therapeutic and Detoxifying Enzymes

Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new-to-nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state-of-the-art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.     

Polymorphic forms of nylon 3

The crystal structure of nylon 3 was studied, and four crystalline modifications were observed. Modification I, as determined from the x-ray diffraction pattern of drawn fibers, is similar to the α crystal structure of nylon 6. The unit cell is monoclinic; a = 9.33 Å, b = 4.78 Å, (fiber identity period), c = 8.73 Å, and β = 60°. The theoretical density for nylon 3 with four monomeric units in the unit cell is 1.39 g/cm³, and the observed density is 1.33 g/cm³. The space group is P2₁. The nylon 3 chains are in the extended planar zigzag conformation. Although other odd-numbered nylon form triclinic or pseudohexagonal crystals when oriented, drawn nylon 3 crystals are monoclinic. In addition to modification I, modifications II, III, and IV were studied. Lattice spacings of modifications II and III are equal to those of modification I. However x-ray diffraction intensities are different. Infrared spectra of those forms indicate an extended planar zigzag conformation of the chains. Modification IV is thought to correspond to the so-called smectic hexagonal form. No γ crystals were found, and it appears that polyamide chains with short sequences of methylene groups cannot form crystals of this type.     

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons

Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho- and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.     

Characterization of the CYP3A4 active site by homology modeling

Human microsomal cytochrome P450s participate in drug metabolism and detoxification. Among them, CYP3A4 is the most important isoform for drug-drug interactions. To gain a better understanding of the active site, a homology model of CYP3A4 was constructed based on the crystallographic coordinates of mammalian CYP2C5. The putative active site is much larger than that of CYP2C5 and is divided into three parts (i.e. a proximal and two distal sites from the heme). Most residues reported to be important for ligand-binding are located in the active site of the model. Moreover, some inhibitors (paclitaxel etc.) docked into the model have complementary shapes to the pocket. Pharmacophore docking of 14 substrates was also performed using Ph4Dock of MOE. Calculated interaction energies showed a moderate correlation with the logarithm of apparent K(m) values. These results suggest that this model is reliable enough to be used in the design of compounds for removing undesirable CYP3A4 inhibition.     

High Levels of Glucose Induces a Dose-Dependent Apoptosis in Human Periodontal Ligament Fibroblasts by Activating Caspase-3 Signaling Pathway

Periodontitis is one of the main complications of diabetes mellitus and many researches have been done on the relationship between periodontitis and diabetes mellitus, but too much are still unclear, especially the mechanisms by which high glucose induces damage of periodontal ligament fibroblasts. So in this study, we investigated the effects of different concentration of high glucose on apoptosis in human periodontal ligament fibroblasts and the possible mechanisms involved. Human periodontal ligament fibroblasts were cultured and subjected to glucose of different concentration (5.5, 15, 25, and 35 mM) for 24 h. Apoptosis was studied by flow cytometry, caspase assays, fluorescent real-time PCR and Western blot. We also determined Fas/FasL expression was by Western blot. The application of different concentration of high glucose induced a concentration-dependent increase of apoptosis and the activity of caspase-3 in cultured human periodontal ligament fibroblasts. Furthermore, inhibitor of caspase-3 could prevent the high-glucose-induced apoptosis in human periodontal ligament fibroblasts. Protein levels of Fas and FasL remained unchanged. These data indicate that high glucose induces a concentration- and caspase-3-dependent increase of apoptosis in cultured human periodontal ligament fibroblasts in vitro. Activation of caspase-3 caused by high glucose is independent of Fas/FasL signaling pathways system. These results suggest a novel mechanism for the regulation of human periodontal ligament fibroblasts apoptosis by high glucose.     

Effect of CYP3A4 on liver injury induced by triptolide

Triptolide (TP), one of the main bioactive diterpenes of the herbal medicine Tripterygium wilfordii Hook F, is used for the treatment of autoimmune diseases in the clinic and is accompanied by severe hepatotoxicity. CYP3A4 has been reported to be responsible for TP metabolism, but the mechanism remains unclear. The present study applied a UPLC–QTOF–MS-based metabolomics analysis to characterize the effect of CYP3A4 on TP-induced hepatotoxicity. The metabolites carnitines, lysophosphatidylcholines (LPCs) and a serious of amino acids were found to be closely related to liver damage indexes in TP-treated female mice. Metabolomics analysis further revealed that the CYP3A4 inducer dexamethasone improved the level of LPCs and amino acids, and defended against oxidative stress. On the contrary, pretreatment with the CYP3A4 inhibitor ketoconazole increased liver damage with most metabolites being markedly altered, especially carnitines. Among these metabolites, except for LPC18:2, LPC20:1 and arginine, dexamethasone and ketoconazole both affected oxidative stress induced by TP. The current study provides new mechanistic insights into the metabolic alterations, leading to understanding of the role of CYP3A4 in hepatotoxicity induced by TP.     

Mechanistic Insights into the Regio- and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1

The hydroxylation of nonreactive C−H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio- and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi-scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio- and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1β, 2β and 19 can access the catalytic center, with the intrinsic reactivity 2β>1β>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s.     

Oligomerization Profile of Human Transthyretin Variants with Distinct Amyloidogenicity

One of the molecular hallmarks of amyloidoses is ordered protein aggregation involving the initial formation of soluble protein oligomers that eventually grow into insoluble fibrils. The identification and characterization of molecular species critical for amyloid fibril formation and disease development have been the focus of intense analysis in the literature. Here, using photo-induced cross-linking of unmodified proteins (PICUP), we studied the early stages of oligomerization of human transthyretin (TTR), a plasma protein involved in amyloid diseases (ATTR amyloidosis) with multiple clinical manifestations. Upon comparison, the oligomerization processes of wild-type TTR (TTRwt) and several TTR variants (TTRV30M, TTRL55P, and TTRT119M) clearly show distinct oligomerization kinetics for the amyloidogenic variants but a similar oligomerization mechanism. The oligomerization kinetics of the TTR amyloidogenic variants under analysis showed a good correlation with their amyloidogenic potential, with the most amyloidogenic variants aggregating faster (TTRL55P > TTRV30M > TTRwt). Moreover, the early stage oligomerization mechanism for these variants involves stepwise addition of monomeric units to the growing oligomer. A completely different behavior was observed for the nonamyloidogenic TTRT119M variant, which does not form oligomers in the same acidic conditions and even for longer incubation times. Thorough characterization of the initial steps of TTR oligomerization is critical for better understanding the origin of ATTR cytotoxicity and developing novel therapeutic strategies for the treatment of ATTR amyloidosis.     

Molecular Docking of 3-Methylindole-containing Drugs Binding into CYP3A4

Drugs SPD-304(6,7-dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]- amino}-ethyl)-amino]-methyl}-chromen-4-one) and zafirlukast contain a common structural element of 3-substituted indole moiety which closely relates to a dehydrogenated reaction catalyzed by cytochrome P450s(CYPs). It was reported that the dehydrogenation can produce a reactive electrophilic intermediate which cause toxicities and inactivate CYPs. Drug L-745,870(3-{[4-(4-chlorophenyl)piperazin-1-yl]-methyl}-1H-pyrrolo- 2,3-β-pyridine) might have similar effect since it contains the same structural element. We used molecular docking approach combined with molecular dynamics(MD) simulation to model three-dimensional(3D) complex structures of SPD-304, zafirlukast and L-745,870 into CYP3A4, respectively. The results show that these three drugs can stably bind into the active site and the 3-methylene carbons of the drugs keep a reasonable reactive distance from the heme iron. The complex structure of SPD-304-CYP3A4 is in agreement with experimental data. For zafirlukast, the calculation results indicate that 3-methylene carbon might be the dehydrogenation reaction site. Docking model of L-745,870-CYP3A4 shows a potential possibility of L-745,870 dehydrogenated by CYP3A4 at 3-methylene carbon which is in agreement with experiment in vivo. In addition, residues in the phenylalanine cluster as well as S119 and R212 play a critical role in the ligands binding based on our calculations. The docking models could provide some clues to understand the metabolic mechanism of the drugs by CYP3A4.     

Enzymatic Production of Ferulic Acid from Defatted Rice Bran by Using a Combination of Bacterial Enzymes

Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-l-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.     

Polymorphic transitions of sodium-magnesium trimetaphosphate NaMg(PO3)3

The existance of the compound NaMg(PO₃)₃, which is formed in the binary system NaPO₃-Mg(PO₃)₂ at the molar ratio of initial metaphosphates 11, has been confirmed. The polymorphism and the way of the formation of this compound have been examined.

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Zusammenfassung Es wurde die Existenz der im binären System NaPO₃-Mg(PO₃)₂ bei einem 11 Molverhältnis der Ausgangsmetaphosphate gebildeten Verbindung NaMg(PO₃)₃ nachgewiesen. Außerdem wurde die Polymorphie und der Entstehungsweg dieser Verbindung untersucht.

     

胶束电动毛细管色谱分析间接紫外吸收检测人体血小板活化因子

研究了人体血小板活化因子中1-O-十六烷基乙酰甘油磷酰胆碱与1-O-八烷基乙酰甘油磷酰胆碱的胶束电动毛细管色谱分离条件与间接紫外吸收检测条件。首次采用高效毛细管电泳法成功地分析了人体血小板活化因子中两种主要磷酰胆碱的含量。     

Constituents of Zingiber aromaticum and their CYP3A4 and CYP2D6 inhibitory activity

A new sesquiterpene 2,9-humuladien-6-ol-8-one (1) was isolated from methanol extract of Zingiber aromaticum, along with 15 known compounds. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by cytochrome P450 3A4 (CYP3A4) and CYP2D6.