Isolation, Substrate Specificity, and Subunit Characterization of the Xylanosomes Produced by Oerskovia turbata JCM 3160.

This work aims at functional studies of the multienzyme complexes produced by Oerskovia turbata JCM 3160 and reveal of their subunit structures. The multienzyme complexes were isolated, enzymatic assayed, the whole genome sequence was determined in fine scale, and the subunit structure was identified by Maldi-TOF mass spectrometry. The isolated multienzyme complexes here show similar particle size with the xylanosomes produced by Cellulosimicrobium cellulans F16, have at least two conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions. This is the first report of the enzymatic activities and subunit structures of xylanosome produced by Oerskovia turbata, providing insights into its diverse capability as well as degrading bias on hemicelluloses.

Isolation and subunit structure of the xylanosome complex produced by Actinotalea fermentans JCM9966.

Xylanosomes, also known as hemicellulosomes, are hemicellulose-degrading nano-scale multienzyme complexes produced by some Firmicutes, Actinobacteria, and Fungi. Here we report the isolation of the MECs produced by Actinotalea fermentas JCM9966, as well as the functional studies and subunit structure revealed by proteomic identifications. The isolated MECs here shows similar particle size with the xylanosomes produced by C. cellulans F16, have several conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions, indicating diverse capability as well as bias in degrading hemicelluloses.

Xylanosomes produced by Cellulosimicrobium cellulans F16 were diverse in size, but resembled in subunit composition.

The hemicellulolytic enzyme system produced by Cellulosimicrobium cellulans strain F16 was resolved by ultracentrifugation and size exclusion chromatography. The particle size and molecular weight were determined by both dynamic light scattering and negative stain electron microscopy. The results showed that xylanosomes produced by strain F16 were found to have an apparent sedimentation coefficient of 28 S, were diverse in size (18-70 nm), molecular weight (11-78 MDa) and morphology, but resembled in subunit composition (SDS-PAGE and proteomic results). It is proposed that particles of 22 nm may be the basic unit, while 43 nm and 60 nm particles observed may be dimer and trimer of the basic unit, or xylanosomes with smaller size might be degradation products of larger size xylanosomes. Moreover, such xylanosomes are also found to have strong binding affinity toward water-insoluble substrates such as Avicel, birchwood xylan, and corn cob.

In vitro characterization of the glucuronidation pathways of licochalcone A mediated by human UDP-glucuronosyltransferases.

This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM). HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM. LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4'-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4'-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4'-O-glucuronide. UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4'-O-glucuronidation.

Effects of Different Carbon Sources on Enzyme Production and Ultrastructure of Cellulosimicrobium cellulans.

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

Characterization and structure-activity relationship studies of flavonoids as inhibitors against human carboxylesterase 2.

Human carboxylesterases (hCEs) are key enzymes from the serine hydrolase superfamily. Among all identified hCEs, human carboxylesterase 2 (hCE2) plays crucial roles in the metabolic activation of ester drugs including irinotecan and flutamide. Selective and potent hCE2 inhibitors could be used to alleviate the toxicity induced by hCE2-substrate drugs. In this study, more than fifty flavonoids were collected to assay their inhibitory effects against hCE2 using a fluorescence-based method. The results demonstrated that C3 and C6 hydroxy groups were essential for hCE2 inhibition, while O-glycosylation or C-glycosylation would lead to the loss of hCE2 inhibition. Among all tested flavonoids, 5,6-dihydroxyflavone displayed the most potent inhibitory effect against hCE2 with the IC50 value of 3.50 µM. The inhibition mechanism of 5,6-dihydroxyflavone was further investigated by both experimental and docking simulations. All these findings are very helpful for the medicinal chemists to design and develop more potent and highly selective flavonoid-type hCE2 inhibitors.

Species of family Promicromonosporaceae and family Cellulomonadeceae that produce cellulosome-like multiprotein complexes.

OBJECTIVES: To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular ß-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights. RESULTS: Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular ß-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit. CONCLUSION: This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular ß-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.

Assessment of the inhibitory effects of pyrethroids against human carboxylesterases.

Pyrethroids are broad-spectrum insecticides that widely used in many countries, while humans may be exposed to these toxins by drinking or eating pesticide-contaminated foods. This study aimed to investigate the inhibitory effects of six commonly used pyrethroids against two major human carboxylesterases (CES) including CES1 and CES2. Three optical probe substrates for CES1 (DME, BMBT and DMCB) and a fluorescent probe substrate for CES2 (DDAB) were used to characterize the inhibitory effects of these pyrethroids. The results demonstrated that most of the tested pyrethroids showed moderate to weak inhibitory effects against both CES1 and CES2, but deltamethrin displayed strong inhibition towards CES1. The IC50 values of deltamethrin against CES1-mediated BMBT, DME, and DMCB hydrolysis were determined as 1.58µM, 2.39µM, and 3.3µM, respectively. Moreover, deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in living cells. Further investigation revealed that deltamethrin inhibited CES1-mediated BMBT hydrolysis via competitive manner but noncompetitively inhibited DME or DMCB hydrolysis. The inhibition behaviors of deltamethrin against CES1 were also studied by molecular docking simulation. The results demonstrated that CES1 had at least two different ligand-binding sites, one was the DME site and another was the BMBT site which was identical to the binding site of deltamethrin. In summary, deltamethrin was a strong reversible inhibitor against CES1 and it could tightly bind on CES1 at the same ligand-binding site as BMBT. These findings are helpful for the deep understanding of the interactions between xenobiotics and CES1.

An Optimized Two-Photon Fluorescent Probe for Biological Sensing and Imaging of Catechol-O-Methyltransferase.

A practical two-photon fluorescent probe was developed for highly sensitive and selective sensing of the activities of catechol-O-methyltransferase (COMT) in complex biological samples. To this end, a series of 3-substituted 7,8-dihydroxycoumarins were designed and synthesized. Among them, 3-BTD displayed the best combination of selectivity, sensitivity, reactivity, and fluorescence response following COMT-catalyzed 8-O-methylation. The newly developed two-photon fluorescent probe 3-BTD can be used for determining the activities of COMT in complex biological samples and bio-imaging of endogenous COMT in living cells and tissue slices with good cell permeability, low cytotoxicity, and high imaging resolution. All these findings suggest that 3-BTD holds great promise for developing therapeutic molecules that target COMT, as well as for exploring COMT-associated biological processes and its biological functions in living systems. Furthermore, the strategy also sheds new light on the development of fluorescent probes for other conjugative enzymes.

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase.

OBJECTIVES: To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin. RESULTS: Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-L-rhamnosidase activity while the ß-D-glucosidase activity was abrogated. The addition of Mg(2+), Fe(2+) and Co(2+) was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and (1)H-NMR. CONCLUSION: A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-L-rhamnosidase activity of naringinase and blocked its ß-D-glucosidase activity.

[The relationship between catechol O-methyltransferase and diseases].

Catechol O-methyltransferase (COMT), one of the endogenous phase II metabolizing enzymes, expressed by chromosome 22. COMT catalyzes the transfer of a methyl group from common methyl donor S-adenosyl-L-methionine(Ado Met or SAM) to one of the catechol hydroxyls. COMT participates in the metabolism of many catechols in vivo, e.g. dopamine, epinephrine, noradrenaline, estradiol. Furthermore COMT also plays important roles in the metabolism of xenobiotic catechols from food and drug. COMT play a critical role in the management of catechols. Metabolism disorders of COMT can cause many diseases or an increased risk of diseases, e.g. Pakinson diseases, schizophrenia, and breast cancer. In this review, we explains the relationship of COMT and related-diseases through expounding disease caused by the COMT metabolic disorders. Finally, we hope that there will be more effective treatments for the COMT metabolism related diseases.

A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

Enzymatic hydrolysis of 7-xylosyltaxanes by an extracellular xylosidase from Cellulosimicrobium cellulans.

OBJECTIVES: To find extracellular biocatalysts that can specifically and efficiently remove the C-7 xylosyl group from 7-xylosyltaxanes. RESULTS: A Cellulosimicrobium cellulans strain F16 that can remove the C-7 xylosyl group from 7-xylosyltaxanes was isolated from the root soil of an old Taxus yunnanensis tree. Using corn cob as sole carbon source, the maximum 7-xylosyl-10-deacetylpaclitaxel ß-xylosidase activity of 9.6 U l(-1) was achieved. The ß-xylosidase could be trapped by a ceramic tubular membrane (pore size 50 nm), and exhibited an apparent molecular weight much greater than 500 kDa. Under optimized conditions, 3.75 l cell-free culture medium transformed 2 grams 7-xylosyltaxane mixtures to their corresponding aglycones within 3 h, with a conversion >98%. CONCLUSION: This is the first report that C. cellulans can produce extracellular ß-xylosidases capable of removing the C-7 xylosyl group from 7-xylosyltaxanes.

Discovery and Characterization of the Key Constituents in Ginkgo biloba Leaf Extract With Potent Inhibitory Effects on Human UDP-Glucuronosyltransferase 1A1.

Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 µM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the K i values ranging from 0.07 to 0.74 µM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.

Isolation and subunit compositions of the xylanosome complexes produced by Cellulosimicrobium species.

Cellulosimicrobium cellulans, which is type species of the genus Cellulosimicrobium, produces xylanase predominant nanoscale multienzyme complexes, i.e., xylanosomes, when grown on water-insoluble polysaccharides. Here, we report on the isolation of similar multienzyme complexes (MECs) produced by two other species in genus Cellulosimicrobium (Cellulosimicrobium funkei and Cellulosimicrobium terreum). Functional studies and subunit structure identifications using genomic sequencing and proteomic techniques were also performed. When compared with the xylanosomes produced by C. cellulans F16, the isolated MECs showed a larger particle size and shared at least three conserved multidomain proteins. In addition, they also exhibited different enzymatic activities and subunit compositions, which indicates diverse capability and strategies in degrading hemicelluloses.

Chemical Probes for Human UDP-Glucuronosyltransferases: A Comprehensive Review.

UGTs play crucial roles in the metabolism and detoxification of both endogenous and xenobiotic compounds. The key roles of UGTs in human health have garnered great interest in the design and development of specific probes for human UGTs. However, in contrast to other human enzymes, the probe substrates for human UGTs are rarely reported, owing to the highly overlapping substrate specificities of UGTs and the lack of the integrated crystal structures of UGTs. Over the past decades, many efforts are made to develop specific probe substrates for UGTs and use them in both basic research and drug discovery. This review focuses on recent progress in the development of probe substrates for UGTs and their biomedical applications. A long list of chemical probes for UGTs, including non-fluorescent and fluorescent probes along with their structural information and kinetic parameters, are prepared and analyzed. Additionally, challenges and future directions in this field are highlighted in the final section. All information and knowledge presented in this review provide practical tools/methods for measuring UGT activities in complex biological samples, which will be very helpful for rapid screening and characterization of UGT modulators, and for exploring the relevance of UGT enzymes to human diseases.

Natural constituents of St. John's Wort inhibit the proteolytic activity of human thrombin.

Thrombin, a multifunctional serine protease responsible for the proteolytic hydrolysis of soluble fibrinogen, plays a pivotal role in the blood coagulation cascade. Currently, thrombin inhibitor therapy has been recognized as an effective therapeutic strategy for the prevention and treatment of thrombotic diseases. In this study, the inhibitory effects of natural constituents in St. John's Wort against human thrombin are carefully investigated by a fluorescence-based biochemical assay. The results clearly demonstrate that most of naphthodianthrones, flavonoids and biflavones exhibit strong to moderate inhibition on human thrombin. Among all tested compounds, hypericin shows the most potent inhibitory capability against thrombin, with the IC50 value of 3.00 µM. Further investigation on inhibition kinetics demonstrates that hypericin is a potent and reversible inhibitor against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki value of 2.58 µM. Inhibition kinetic analyses demonstrate that hypericin inhibits thrombin-mediated Z-GGRAMC acetate hydrolysis in a mixed manner, which agrees well with the results from docking simulations that hypericin can bind on both catalytic cavity and anion binding exosites. All these findings suggest that hypericin is a natural thrombin inhibitor with a unique dianthrone skeleton, which can be used as a good candidate to develop novel thrombin inhibitors with improved properties.

Inhibition of human carboxylesterases by ginsenosides: structure-activity relationships and inhibitory mechanism.

BACKGROUND: Human carboxylesterases (hCES) are key serine hydrolases responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters. Although it has been reported that some ginsenosides can modulate the activities of various enzymes, the inhibitory effects of ginsenosides on hCES have not been well-investigated. METHODS: In this study, more than 20 ginsenosides were collected and their inhibitory effects on hCES1A and hCES2A were assayed using the highly specific fluorescent probe substrates for each isoenzyme. Molecular docking simulations were also performed to investigate the interactions between ginsenosides and hCES. RESULTS: Among all tested ginsenosides, Dammarenediol II (DM) and 20S-O-ß-(d-glucosyl)-dammarenediol II (DMG) displayed potent inhibition against both hCES1A and hCES2A, while protopanaxadiol (PPD) and protopanaxatriol (PPT) exhibited strong inhibition on hCES2A and high selectivity over hCES1A. Introduction of O-glycosyl groups at the core skeleton decreased hCES inhibition activity, while the hydroxyl groups at different sites might also effect hCES inhibition. Inhibition kinetic analyses demonstrated that DM and DMG functioned as competitive inhibitors against hCES1A-mediated d-luciferin methyl ester (DME) hydrolysis. In contrast, DM, DMG, PPD and PPT inhibit hCES2A-mediated fluorescein diacetate (FD) hydrolysis via a mixed manner. CONCLUSION: The structure-inhibition relationships of ginsenosides as hCES inhibitors was investigated for the first time. Our results revealed that DM and DMG were potent inhibitors against both hCES1A and hCES2A, while PPD and PPT were selective and strong inhibitors against hCES2A.

Anthraquinones from Cassiae semen as thrombin inhibitors: in vitro and in silico studies.

Thrombin inhibitor therapy is one of the most effective therapeutic strategies for the prevention and treatment of cardiovascular and thrombotic diseases. Although several marketed direct thrombin inhibitors (DTIs) have been widely used in clinic, the potentially serious complications of these DTIs prompted the researchers to find more DTIs with improved safety profiles. Herein, we report that natural anthraquinones in Cassiae semen (the seed of Cassia obtusifolia L. or C. tora L.), including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin, display strong to moderate inhibition on human thrombin, with the IC50 values ranging from 9.08 µM to 27.88 µM. Further investigation on the inhibition kinetics demonstrates that these anthraquinones are mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, while obtusifolin and aurantio-obtusin show strong thrombin inhibition capacity, with the Ki values of 9.63 µM and 10.30 µM, respectively. Docking simulations demonstrate that both obtusifolin and aurantio-obtusin can simultaneously bind on the catalytic cavity and the two anion binding exosites (ABE1 and ABE2), while the hydroxyl group at the C-7 site and the methoxyl group at the C-8 site can create key interactions with the amino acids surrounding the catalytic cavity via hydrogen bonding. All these findings suggest that obtusifolin and aurantio-obtusin are strong thrombin inhibitors possessing a unique anthraquinone skeleton, and could be used as lead compounds for the development of new thrombin inhibitors with improved properties.

Inhibition of human carboxylesterases by magnolol: Kinetic analyses and mechanism.

Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.