Esterase Assay Research Articles

Methanol extract of Antrodia cinnamomea mycelia induces phenotypic and functional differentiation of HL60 into monocyte-like cells via an ERK/CEBP-β signaling pathway

Antrodia cinnamomea (named as Niu-chang-chih), a well-known Taiwanese folk medicinal mushroom, has a spectrum of biological activities, especially with anti-tumor property. This study was carried out for the first time to examine the potential role and the underlying mechanisms of A. cinnamomea in the differentiation of human leukemia HL60 cells. We found that the methanol extract of liquid cultured mycelia of A. cinnamomea (MEMAC) inhibited proliferation and induced G1-phase cell cycle arrest in HL60 cells. MEMAC could induce differentiation of HL60 cells into the monocytic lineage, as evaluated by the morphological change, nitroblue tetrazolium reduction assay, non-specific esterase assay, and expression of CD14 and CD11b surface antigens. In addition, MEMAC activated the extracellular signal-regulated kinase (ERK) pathway and increased CCAAT/enhancer-binding protein β (C/EBPβ) expression. Reverse transcriptase polymerase chain reaction analysis showed that MEMAC upregulated the expression of C/EBPβ and CD14 mRNA in HL60 cells. DNA affinity precipitation assay and chromatin immunoprecipitation analyses indicated that MEMAC enhanced the direct binding of C/EBPβ to its response element located at upstream of the CD14 promoter. Furthermore, inhibiting ERK pathway activation with PD98059 markedly blocked MEMAC-induced HL60 monocytic differentiation. Consistently, the MEMAC-mediated upregulation of C/EBPβ and CD14 was also suppressed by PD98059. These findings demonstrate that MEMAC-induced HL60 cell monocytic differentiation is via the activating ERK signaling pathway, and downstream upregulating the transcription factor C/EBPβ and differentiation marker CD14 gene, suggesting that MEMAC might be a potential differentiation-inducing agent for treatment of leukemia.

Assessing phytoplankton cell lysis rate in Lake Taihu by esterase assay

本研究从2009年8月至2010年10月,每月采集太湖3个不同富营养化湖区水样,运用酯酶活性法,测定了颗粒态酯酶、溶解性酯酶活性以及酯酶衰变周期,估算了太湖浮游植物细胞裂解速率.研究结果表明,太湖颗粒态酯酶活性为0.58~35.15 nmol FDA/(L.h),溶解性酯酶活性为0.55~7.59 nmol FDA/(L.h),酯酶衰变周期为7~75 h,细胞裂解速率为0.02~0.77 d^-1,三个采样点细胞裂解速率没有显著差异.颗粒态酯酶活性与叶绿素a浓度之间具有显著的线性关系,说明运用酯酶活性法估算太湖浮游植物细胞裂解速率是可行.此外,叶绿素a浓度与温度变化趋势基本一致,梅梁湾和湖心叶绿素a浓度具有显著差异.贡湖湾叶绿素a浓度与细胞裂解速率之间具有显著的反比例关系,说明细胞裂解速率也是影响太湖藻类生物量的重要因素.;Phytoplankton lysis rate in three different eutrophic regions of Lake Taihu was measured by the activities of particle and dissolved esterase, as well as the decay rate of the latter, from August 2009 to October 2010. The present results showed that the particulate, dissolved esterase activity, half life time of dissolved esterase, and cell lysis rates ranged from 0.58-35.15 nmol FDA/(L·h), 0.55-7.59 nmol FDA/(L·h), 7-75 h, and 0.02-0.77 d^-1, respectively. No significant differences were found in the cell lysis rates among the three sampling sites. The significant linear relationship between Chl.a concentration and particulate esterase activities suggests that it is feasible to assess phytoplankton cell lysis rate in Lake Taihu by the esterase assay. Furthermore, Chl.a concentration showed the same pattern with water temperature, a significant difference was observed in Chl.a concentration between Meiliang Bay and lake centre. The strong, negative correlation between Chl.a concentration and lysis rate in Gonghu Bay suggests that phytoplankton biomass was affected by the lysis rate in Lake Taihu.

Carbonic anhydrase inhibitors: in vitro inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by flavonoids

A series of flavonoids, such as quercetin, catechin, apigenin, luteolin, morin, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA). The compounds were tested against four α-CA isozymes purified from human and bovine (hCA I, hCA II, bCA III, hCA IV) tissues. The four isozymes showed quite diverse inhibition profiles with these compounds. The flavonoids inhibited hCA I with KI-s in the range of 2.2–12.8 μM, hCA II with KI-s in the range of 0.74–6.2 μM, bCA III with KI-s in the range of 2.2–21.3 μM, and hCA IV with inhibition constants in the range of 4.4–15.7, with an esterase assay using 4-nitrophenyl acetate as substrate. Some simple phenols/sulfonamides were also investigated as standard inhibitors. The flavonoids incorporate phenol moieties which inhibit these CAs through a diverse, not yet determined inhibition mechanism, compared to classic inhibitors such as the sulfonamide/sulfamate ones.

In Vitro Inhibition of Human Carbonic Anhydrase I and II Isozymes with Natural Phenolic Compounds

Inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II with some natural phenolic derivatives was investigated using the esterase assay with 4-nitrophenyl acetate as substrate. Resveratrol, catechin, silymarin, dobutamin, and curcumin showed K(I) values in the range of 4.47-9.47 mm for hCA I and of 2.86-7.44 μm against hCA II, respectively. These natural product phenols were generally competitive inhibitors with 4-nitrophenylacetate as substrate. Some natural phenols investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms that have not been yet assayed for their interactions with such agents.

Legionella nagasakiensis sp. nov., isolated from water samples and from a patient with pneumonia

A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, β-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

BIOCHEMICAL MARKERS: A USEFUL TOOL FOR ASSESSING GENETIC DIVERSITY IN JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAM.)

Using polyacrylamide gel electrophoresis, peroxidase and esterase isozymes were investigated from leaf tissue samples of 44 selected (superior) jackfruit genotypes. These selected genotypes were collected following jackfruit descriptor 2000, after surveying 1500 trees in the districts of Nadia, 24 Parganas (N) and Coochbehar in West Bengal. The selected genotypes were analyzed for isozyme variation for peroxidase and esterase. Polymorphism was observed in both the enzyme systems studied, where several bands were found showing similarities and dissimilarities among the selected types. In the peroxidase assay, 7 loci were identified among the 44 genotypes. The relative mobility (R m ) values of the loci ranged between 0.29 and 0.50. Whereas in the esterase assay, 6 loci were identified and the relative mobility (R m ) values of the loci ranged between 0.22 and 0.65. Moreover, it was observed that genotypes collected from nearby locations did not show similar banding patterns, indicating that forces other than geographical origin, such as exchange of genetic stocks, genetic drift, spontaneous variation, natural and artificial selection, cross pollination and seedling origin of the plant are responsible for the genetic diversity.

Structure Based Inhibition of Mitochondrial Aldehyde Dehydrogenase (ALDH2) Activity

Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme that participates in multiple metabolic pathways, including the oxidation of toxic biogenic and environmental aldehydes. Our laboratory is interested in developing novel and selective ALDH2 inhibitors. We recently identified and studied three distinct classes of small molecules based on their ability to inhibit ALDH2 activity (esterase, dehydrogenase, and both). These inhibitors were identified by virtual screen which consisted of three steps: 1) docking 800,000 ChemBridge molecules to the substrate cavity in ALDH2; 2) scoring the receptor-ligand complexes while computing the binding affinity; and 3) ranking the top 1,000 compounds using GlideScore to determine the top 250 compounds for study. From this list, 112 compounds were selected for purchase and screened for inhibitory activity at 50 uM. 19 compounds were selected for further evaluation based on their ability to inhibit hALDH2 propionaldehyde oxidation by more than 60%. IC50 values for these compounds were determined using both the dehydrogenase and esterase assays. We selected 4 compounds with IC50 values less than 20uM for further kinetic and structural studies: I32, I72, I76, and I78. These inhibitors show a competitive inhibition pattern toward varied NAD+ concentrations and either uncompetitive or noncompetitive inhibition towards varied priopionaldehyde concentrations. Based on their structural similarity and x-ray crystallography structures obtained with these compounds bound to the enzyme, fragments were then chosen for studying the mechanism by which they inhibit ALDH2. The next step is to solve the structures of these fragments bound to ALDH2 and develop SAR data for fragment based compound development that will enhance their specificity for and inhibition of ALDH2. This work was supported by NIH R01-AA18123 and NIH R01-AA18123S1.

Biochemical studies on malathion resistance, inheritance and association of carboxylesterase activity in brown planthopper, Nilaparvata lugens complex in Peninsular Malaysia

Abstract Two sympatric populations of brown planthopper (BPH), one from rice and the other from Leersia hexandra were collected from each of five locations in Malaysia. All the tested malathion‐resistant individuals of the rice BPH population and F1 generation (cross between malathion‐resistant [usually caught on rice] and malathion‐susceptible [usually caught on Leersia]) showed high esterase activity, while all malathion‐susceptible individuals on L. hexandra showed low esterase activity. In the F2 generation, all the individuals tested against malathion were approximately 75% resistant and 25% susceptible and the inheritance pattern of esterase activity (high and low esterase activity) segregated in the same manner to a 3: 1 ratio. This confirms that resistance to malathion is mono‐factorial and inheritance pattern of esterase activity is also linked to malathion resistance. Carboxylesterase or total esterase activity in BPH is inherited in a simple Mendelian fashion that is encoded by a single dominant gene. For the total esterase assay, average esterase activity levels in the rice‐infesting population ranged from 17.64 to 19.37 nmoles 1‐napthol/mg protein while that in the Leersia‐infesting population ranged from 5.29 to 6.11 nmoles 1‐napthol/mg protein. In terms of esterase activity, the two sympatric Nilaparvata lugens populations separated into two distinct groups. Results based on the tube color intensity test showed 96% and 98% resistant and susceptible individuals were present in the rice‐ and Leersia‐infesting populations, respectively. In a filter paper test, the rice‐infesting population had 94% with high esterase activity while the Leersia‐infesting population had 96% with low esterase activity.

Cell Lysis of Cyanobacteria and Its Implications for Nutrient Dynamics

AbstractThe dynamics of nutrients, such as phosphorus, nitrogen, and carbohydrates, during cyanobacteria cell lysis was investigated under darkness incubation in the laboratory. The cell lysis rate of cyanobacteria sampled from Lake Taihu was measured using an esterase assay. Based on particulate esterase activity, the calculated cyanobacteria lysis rate was 0.094 d–1. During 30 days of darkness incubation, Chlorophyll a concentration decreased from 56 μg L–1 to 2.0 μg L–1. Parallel to this, total particulate carbohydrate concentration decreased rapidly. The fluctuation of dissolved organic carbon concentration was a function of the production of non‐carbohydrate by cyanobacteria and the decomposition of carbohydrate by bacteria. Total dissolved carbohydrates and dissolved polysaccharides concentrations showed a similar pattern, declining at the beginning of the experiment and keeping relatively stable, thereafter. In contrast, the concentration of dissolved monosaccharides remained constant during the entire process. The concentrations of NH4+ and PO43– increased at the early stage, and then decreased afterwards. A gradual decrease in NO3– concentration after day 8 indicated that anaerobic conditions might be produced during the cell lysis process. The present results demonstrated cyanobacteria cell lysis has a big influence on the nutrient status of the surrounding water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Reduction of plasma granzyme A correlates with severity of sepsis in burn patients

The risk of mortality is high in burn patients and correlates with age, burn area extent, and sepsis. Immunosuppression has been reported to occur after severe burn. Cytotoxic cells possess specialized granules containing perforin and a group of serine proteases (granzymes). Granzyme A is a serine protease constitutively expressed by γδ and NK cells, in agreement with their functional cytolytic potential. In vitro studies have shown that GrA may be released extracellularly during cytotoxic cell degranulation, indicating the activation of cytotoxic cells. The aim of our study was to determine plasma GrA activity in burned patients and to verify if decreased GrA levels were associated with poor prognosis. Specific GrA activity was tested in the plasma of burned and healthy subjects by esterase assay. Plasma GrA was significantly decreased in septic rather than in nonseptic burn patients and in healthy subjects ( p < 0.05 and p < 0.001, respectively). At day 3 plasma GrA was significantly lower in nonsurvivor than in survivor septic patients ( p < 0.05). The value of 91 mOD showed a sensitivity of 100% and a specificity of 84% in differentiating survivor from nonsurvivor septic patients. Because this is a retrospective study, Granzyme A is not a confirmed predictor of septic outcome after burn, but its determination could give useful information about the development and severity of sepsis.

Biosensor analysis of blood esterases for organophosphorus compounds exposure assessment: Approaches to simultaneous determination of several esterases

This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was ∼6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds.

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase

A mammalian family of lipid hydrolases, designated "patatin-like phospholipase domain containing (PNPLA)" recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

Carbonic anhydrase inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with a series of phenol derivatives was investigated by using the esterase assay, with 4-nitrophenyl acetate as substrate. 2,6-Dimethylphenol, 2,6-diisopropylphenol (propofol), 2,6-di- t-butylphenol, butylated hydroxytoluene, butylated hydroxyanisole, vanillin, guaiacol, di(2,6-dimethylphenol), di(2,6-diisopropylphenol), di(2,6-di- t-butylphenol), and acetazolamide showed K I values in the range of 37.5–274.5 μM for hCA I and of 0.29–113.5 μM against hCA II, respectively. All these phenols were non-competitive inhibitors with 4-nitrophenylacetate as substrate. Some antioxidant phenol derivatives investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

Single-step synthesis of 4-nitrophenyl ferulate for spectrophotometric assay of feruloyl esterases

We report here, for the first time, a single-step method for the synthesis of 4-nitrophenyl ferulate (4NPF), a spectrophotometric substrate for the assay of feruloyl esterases by dehydrative coupling of ferulic acid and 4-nitrophenol.

Biochemical and Biological Properties of 4-(3-phenyl-[1,2,4] thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide, a Mechanism-Based Inhibitor of Fatty Acid Amide Hydrolase

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

Dietary supplementation of l-tryptophan mitigates crowding stress and augments the growth in Cirrhinus mrigala fingerlings

A 60 days feeding trial was conducted to study the stress mitigation and hence growth augmenting effect of dietary l-tryptophan during high density group stress in Cirrhinus mrigala fingerlings. Four hundred eighty fingerlings were distributed into eight experimental groups. Each group either of low density group (10 fishes/75 L water) or higher density group (30 fishes/75 L water) was fed with a diet containing either 0, 0.68, 1.36 or 2.72% l-tryptophan in the diet, thus eight experimental groups viz. low density control (LC) (basal feed + 0% l-tryptophan); LT1 (basalfeed + 0.68% l-tryptophan); LT2 (basalfeed + 1.36% l-tryptophan); LT3 (basalfeed + 2.72% l-tryptophan) high density control (HC) (basal feed + 0% l-tryptophan); HT1 (basalfeed + 0.68% l-tryptophan); HT2 (basalfeed + 1.36% l-tryptophan); and HT3 (basalfeed + 2.72% l-tryptophan) were fed at 3% of the body weight with isonitrogenous (34.33 ± 0.23 to 35.81 ± 0.18 CP%) and isocaloric (423.49 ± 1.76 to 425.85 ± 0.31 K.Cal/100 g) purified diets. The possible role of dietary l-tryptophan on stress mitigation was assessed in terms of blood glucose, plasma cortisol, lactate dehydrogenase (LDH), malate dehydrogenase (MDH), alanine amino transferase (ALT), aspartate amino transferase (AST), acetyl choline esterase (AChE) assays, whereas growth was evaluated in terms of weight gain %, specific growth (SGR) and protein efficiency ratio (PER). The LDH, MDH, ALT, AST activities were found to be significantly higher ( p < 0.05) in the control groups and decreasing trend of LDH, MDH, ALT and AST activities were observed with the increasing level of dietary l-tryptophan. However, high density group exhibited significantly higher value ( p < 0.05) for the above parameters than their low density counterpart at similar level of l-tryptophan in the diet. In contrast to above parameters, AChE activity exhibited a reverse trend. In both the stocking densities, l-tryptophan supplemented groups found to have higher ( p < 0.05) growth, SGR and PER. The Plasma cortisol level was found to be significantly higher in the high density group than its low density counterpart. However, gradual supplementation of l-tryptophan in diet significantly reduced the cortisol level both in high density ( Y = − 26.33 x + 219.53, r 2 = 0.95) and low density group ( Y = − 27.57 x + 169.28, r 2 = 0.92). Similar trend was also found in blood glucose. Hence, dietary supplementation of l-tryptophan at a minimum level of 1.36% concomitantly reduced the stress in C. mrigala fingerlings. Though 2.72% dietary tryptophan also reduces the stress but 1.36% level appears to be cost effective.

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the 14C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization–tandem mass spectrometry (ESI–MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Cutinase Inhibition by Means of Insecticidal Organophosphates and Carbamates. 3. Oxidation of Phosphorothionates by Chloroperoxidase from Caldariomyces fumago

Chloroperoxidase (CPO) from Caldariomyces fumago combined with hydrogen peroxide and chloride proved to be most efficient for the transformation of organophosphorothionate pesticides, i.e., chlorpyrifos, chlorpyrifos-methyl, parathion, and parathion-methyl, into their more potent serine esterase inhibiting oxon analogues. Following CPO pre-oxidation steps, the detection limit of a recently described spectrophotometric cutinase assay could be increased by about 2 orders of magnitude as a consequence of increased inhibition rates of the organophosphates. This type of enzymatic oxidation is easier to perform and more efficient, as compared to bromine or N-bromosuccinimide, used for acetylcholine esterase (AChE) assay in water analyses, but is insufficient for complex matrices such as plant sample extracts. The performance of a complete assay, including sample preparation, oxidation, and inhibition, takes about 3 h. Performing oxidations of organophosphorus compounds, two significant anomalies were observed. Upon CPO oxidation, chlorpyrifos-methyl showed a very strong cutinase inhibition as compared to the corresponding oxon standard, and oxidized malathion, contrarily to malaoxon, revealed cutinase inhibition, which however obeyed a reversible reaction mechanism in contrast to the usually irreversible reactions of organophosphates. Except for methomyl, no significant effects of CPO oxidation on the inhibition strength of insecticidal carbamates could be detected. The applicability of the assay was tested with fruit samples spiked with chlorpyrifos at 0.2-0.5 mg/kg, thereby regarding the role of the latter as the pesticide detected most often in fruits. Mean recoveries ranged between 30-50%. An enhanced recovery of 84% was obtained for an apple juice sample spiked with parathion-methyl (0.5 mg/L).

NeuA Sialic Acid O-Acetylesterase Activity Modulates O-Acetylation of Capsular Polysaccharide in Group B Streptococcus

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.