Dual Enzyme Research Articles

Structural Insight into a Membrane Intrinsic Acyltransferase from Chlorobium tepidum.

The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.

Structure and Mechanism of Acetylation by the N-Terminal Dual Enzyme NatA/Naa50 Complex

NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We find that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. These studies have implications for understanding the role of the NatA/Naa50 complex in modulating the majority of the N-terminal acetylome in diverse species.

A novel on-flow mass spectrometry-based dual enzyme assay

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.

A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria.

SummaryNitrate is available to microbes in many environments due to sustained use of inorganic fertilizers on agricultural soils and many bacterial and archaeal lineages have the capacity to express respiratory (Nar) and assimilatory (Nas) nitrate reductases to utilize this abundant respiratory substrate and nutrient for growth. Here, we show that in the denitrifying bacterium Paracoccus denitrificans, NarJ serves as a chaperone for both the anaerobic respiratory nitrate reductase (NarG) and the assimilatory nitrate reductase (NasC), the latter of which is active during both aerobic and anaerobic nitrate assimilation. Bioinformatic analysis suggests that the potential for this previously unrecognized role for NarJ in functional maturation of other cytoplasmic molybdenum‐dependent nitrate reductases may be phylogenetically widespread as many bacteria contain both Nar and Nas systems.

Dual-enzyme-propelled unbounded DNA walking nanomachine for intracellular imaging of lowly expressed microRNA

Despite the progress on the analysis of miRNA either in vitro or in vivo, intracellular imaging of lowly expressed microRNA remains a challenge. Here we develop a novel dual–enzyme–propelled DNA walking nanomachine, which is tailored to accomplish this mission. The nanomachine is constructed with nanoparticles–loaded DNA tracks, on which the targeted miRNA working as a single–foot DNA walker can move autonomously under the catalysis of two cooperative enzymes. Cleavage of the DNA tracks like a “burnt–bridge” mechanism is thereafter triggered, resulting in an amplified fluorescent signal. After the comprehensive study and optimization of the DNA nanomachine, miR–892b, a significantly down–regulated miRNA in breast cancer cells, is selected as a model target. Sensitivity detection in vitro is achieved with a superior detection limit of 4 pM. While being delivered into cells, the DNA nanomachine is available for the imaging of the lowly expressed microRNA, which is totally missing using the conventional fluorescence in situ hybridization (FISH) method. Up–regulation or down–regulation of the miRNA by exogenous regulatory factors can be also well evaluated. This DNA nanomachine provides a competitive approach for the analysis of miRNA, and has the potential to be extended to some other biomolecules.

Dual enzyme activated fluorescein based fluorescent probe

A simple dual analyte fluorescein-based probe (PF3-Glc) was synthesised containing β-glucosidase (β-glc) and hydrogen peroxide (H2O2) trigger units. The presence of β-glc, resulted in fragmentation of the parent molecule releasing glucose and the slightly fluorescent mono-boronate fluorescein (PF3). Subsequently, in the presence of glucose oxidase (GOx), the released glucose was catalytically converted to d-glucono-δ-lactone, which produced H2O2 as a by-product. The GOx-produced H2O2, resulted in classic H2O2-mediated boronate oxidation and the release of the highly emissive fluorophore, fluorescein. This unique cascade reaction lead to an 80-fold increase in fluorescence intensity.

Binding to an Unusual Inactive Kinase Conformation by Highly Selective Inhibitors of Inositol-Requiring Enzyme 1α Kinase-Endoribonuclease

A series of imidazo[1,2-b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.

Improving Photovoltaic and Enzymatic Sensing Performance by Coupling a Core-Shell Au Nanorod@TiO2 Heterostructure with the Bioinspired l-DOPA Polymer.

The photoelectrochemistry (PEC) performance of TiO2 is somewhat limited by its wide band gap and low quantum efficiency, and the innovation of its composite materials provides a promising solution for an improved performance. Herein, a composite of a Au nanorod@TiO2 core-shell nanostructure (AuNR@TiO2) and a melanin-like l-DOPA polymer (PD) is designed and prepared, where the outer layer PD tethered by TiO2-hydroxyl complexation and the AuNR core can intensify the long-wavelength light harvesting, and the AuNR@TiO2 core-shell structure can strengthen the hot-electron transfer to TiO2. The photocurrent of PD/AuNR@TiO2 is 8.4-fold improved versus that of commercial TiO2, and the maximum incident photon-to-electron conversion efficiency reaches 65% in the UV-visible-near-infrared region. In addition, the novel PD/AuNR@TiO2 photocatalyst possesses the advantages of good biocompatibility and stability, which can act as a versatile PEC biosensing platform for providing a biocompatible environment and improving detection sensitivity. Herein, a PEC enzymatic biosensor of glucose is developed on the basis of the immobilization of dual enzyme [glucose oxidase (GOx) and horseradish peroxidase (HRP)] in PD and the signaling strategy of biocatalytic precipitation. In phosphate buffer containing glucose and 4-chloro-1-naphthol, the HRP-catalyzed oxidation of 4-chloro-1-naphthol by GOx-generated H2O2 can form a precipitate on the electrode, by which the decrement of photocurrent intensity is proportional to the common logarithm of glucose concentration. The linear detection range is from 0.05 μM to 10.0 mM glucose, with a limit of detection of 0.01 μM (S/N = 3). Glucose in some human serum samples is analyzed with satisfactory results.

Chemiluminescent dual-enzyme immunoassays capable of simultaneously quantifying carbohydrate antigen 19–9 and carcinoma embryonic antigen in a sample

Using the internal chemiluminescence resonance energy transfer (Inter-CRET) of luminescent dye and the high-energy intermediate formed in 1,1‘-oxalyldiimidazole chemiluminescence (ODI-CL) reaction, we developed for the first time dual-enzyme immunoassays operated with alkaline phosphatase (ALP) and horseradish peroxidase (HRP) for the simultaneous quantifications of carbohydrate antigen 19–9 (CA19-9) and carcinoma embryonic antigen (CEA) in a sample. Fluorescein formed from the ALP enzyme immunoassay emits green light in ODI-CL reaction, while resorufin formed from the HRP enzyme immunoassay emits red light. Green and red CL lights emitted in a detection cell were measured individually with two photomultiplier tubes with an optical filter capable of sensing green or red emission without interferences. The limits of detection for CA 19–9 (0.09 U/ml) and CEA (0.11 ng/ml) determined using the accurate and reproducible dual-enzyme immunoassays were as low as those calculated using the conventional single-enzyme immunoassays capable of sensing CA 19–9 or CEA in a sample. In conclusion, we confirmed that the highly selective dual enzyme immunoassays can be applied as a new analytical method capable of early diagnosing and monitoring colon and pancreatic cancers with the simultaneous analyses of CA 19–9 and CEA.

Mesoporous manganese oxide/manganese ferrite nanopopcorns with dual enzyme mimic activities: A cascade reaction for selective detection of ketoses

Nanomaterials possessing enzyme-like activity have been extensively studied owing to their high stability and tunable catalytic properties. In this work, a simple method has been developed for the synthesis of porous manganese oxide/manganese ferrite (MnOx/MnFe2O4) nanopopcorns (MFNPs) in neutral media. The MFNPs exhibit dual enzymatic activities towards selective oxidation of ketoses followed by H2O2-induced decline of its catalytic activity. MFNPs, with MnFe2O4 as the core material and an outer layer rich in MnOx, were synthesized from ammonium iron(III) citrate and potassium permanganate at 70 °C for 12 h followed by annealing at 300 °C for 6 h. The nanozyme, MFNPs, exhibited oxidase-like activity, which was proved by the oxidation of amplex red (AR) in the presence of dissolved oxygen in the solution, to form fluorescent resorufin. The activity of MFNPs is highly suppressed by H2O2 as a result of its induced dissolution of MnOx. In addition, MFNPs having catalytic activity towards the selective oxidation of ketoses (e.g., fructose) followed by the formation of H2O2. The as-formed H2O2 diminished the catalytic activity of MFNPs for the AR oxidation to form fluorescent resorufin. Upon increasing fructose concentration, the fluorescence of resorufin decreases. Since the MFNPs do not show catalytic activity towards aldose sugars, such as glucose, sucrose, and mannose, the AR/MFNPs probe has high selectivity and sensitivity for detection of fructose with a limit of detection of 32 μM. Our study shows its great potential for quantitation of fructose in honey samples.

Detection of D-glutamate production from the dual Function enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis.

D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen et al., 2016 ). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.

Synthesis and Evaluation of Some Novel 6-Substituted Quinazoline Derivatives as Antitumor Agents

A series of novel 6-substituted quinazoline derivatives were synthesised as epidermal growth factor receptor(EGFR) and Human epidermal growth factor receptor 2 (HER2)inhibitors in our lab. The novel compounds were measured for their dual enzyme inhibition as well as their cytotoxic activity on MCF7 cell line. The results revealed that all the compounds showed inhibition of both enzymes. Compound 5c showed the best inhibitory activity against both enzymes and IC50 of its was 2.6 nM against EGFR kinases and 4.3 nM against HER2 kinases, respectively. Most of the measured compounds showed to antitumor activity on MCF7.

MoO3−x nanodots with dual enzyme mimic activities as multifunctional modulators for amyloid assembly and neurotoxicity

Development of effective inhibitors toward Aβ aggregation and reactive oxygen species (ROS) scavengers are of crucial therapeutic implications for Alzheimer’s disease (AD). Herein, a novel agent with dual enzyme mimic activities has been fabricated as a multifunctional Aβ fibrillation modulator. MoO3−x nanodots were synthesized by pulsed laser ablation (PLA) method in MoS2 nanosheets solutions, which may act directly as numerous fine targets. MoO3−x nanodots showed a uniform and monodispersed morphology, and the tiny dots were around 3–5 nm with a narrow size distribution. Due to the efficient charge transition between Mo5+/Mo6+ on the dots surface, MoO3−x nanodots exhibited excellent catalase and SOD mimic activities, which were adopted to alleviate Aβ-mediated oxidative stress. Moreover, MoO3−x nanodots can efficiently inhibit Aβ aggregation and destabilize the preformed fibrils, and eventually protect neuronal cells from apoptosis induced by Aβ. Taken together, MoO3−x nanodots with multifunctional roles can act as a potential therapeutic strategy for treatment of amyloid induced neurotoxicity.

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica).

BackgroundThe barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioral, and genetic studies. To facilitate further population genetics and genomic studies, we present a reference genome assembly for the European subspecies (H. r. rustica).FindingsAs part of the Genome10K effort on generating high-quality vertebrate genomes (Vertebrate Genomes Project), we have assembled a highly contiguous genome assembly using single molecule real-time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived from both the Nick, Label, Repair, and Stain technology and from the Direct Label and Stain (DLS) technology. As proposed by Bionano, DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of more than 25.95 Mbp.ConclusionsThis high-quality genome assembly represents a valuable resource for future studies of population genetics and genomics in the barn swallow and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies.

Dual Responsive Enzyme Mimicking of Ternary Polyaniline–MnO2–Pd Nanowires and Its Application in Colorimetric Biosensing

In recent years, a significant challenge in material science has been to fabricate novel functional nanomaterials to mimic the function of natural enzymatic systems. Herein, ternary polyaniline (PANi)–MnO2–Pd nanowires (NWs) are synthesized through a two-step redox reaction process. The prepared PANi–MnO2–Pd NWs were explored to exhibit dual responsive enzyme-like efficiencies, e.g., peroxidase and oxidase-like activities. With the assistance of H2O2, the obtained ternary PANi–MnO2–Pd NWs were able to oxidize the typical peroxidase substrate of 3,3′,5,5′-tetramethylbenzidine (TMB) to yield the oxidized TMB with a blue color, exhibiting a much better peroxidase-like activity than the synthesized PANi–MnO2 NWs alone and Pd nanoparticles owing to the interfacial electron transfer effect among the components. In addition, in the absence of H2O2, the prepared PANi–MnO2–Pd NWs could also oxidize TMB to display an excellent oxidase-like activity. Based on the dual responsive enzyme-like characteristic of the obt...

A versatile label-free electrochemical biosensor for circulating tumor DNA based on dual enzyme assisted multiple amplification strategy

A versatile label-free electrochemical biosensor based on dual enzyme assisted multiple amplification strategy was developed for ultrasensitive detection of circulating tumor DNA (ctDNA). The biosensor consists of a triple-helix molecular switch (THMS) as molecular recognition and signal transduction probe, ribonuclease HII (RNase HII) and terminal deoxynucleotidyl transferase (TdT) as dual enzyme assisted multiple amplification accelerator. The presence of target ctDNA could open THMS and trigger RNase HII-assisted homogenous target recycling amplification to produce substantial signal transduction probe (STP). The released STP hybridized with the capture probe immobilized on a gold electrode, then TdT and assistant probe were further employed to fulfill TdT-mediated cascade extension and generate stable DNA dendritic nanostructures. The electroactive methyl blue (MB) was finally used as the signal reporter to realize the multiple electrochemical amplification ctDNA detection as the amount of MB is positively correlated with the target ctDNA. Combined with the efficient recognition capacity of the designed THMS and the excellent multiple amplification ability of RNase HII and TdT, the constructed sensing platform could detect KRAS G12DM with a wide detection range from 0.01 fM to 1 pM, and the limit of detection as low as 2.4 aM. Besides, the platform is capable of detecting ctDNA in biological fluid such as plasma. More importantly, by substituting the loop of THMS with different sequences, this strategy could be conveniently expanded into the detection of other ctDNA, showing promising potential applications in clinical cancer screening and prognosis.

Bioconversion of sucrose to maltooligosaccharides by the synergistic action of amylosucrase and α-amylase

Abstract Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by α-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel α-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from α-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 °C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction.

TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy.

5-Fluorouracil (5-FU) is an antimetabolite and exerts antitumor activity via intracellularly and physiologically complicated metabolic pathways. In this study, we designed a novel small molecule inhibitor, TAS-114, which targets the intercellular metabolism of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addition, TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addition of TAS-114 was retained in the presence of a potent DPD inhibitor containing oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment. Mol Cancer Ther; 17(8); 1683-93. ©2018 AACR.

An allelic series at the KARRIKIN INSENSITIVE2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A.thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable inplanta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging

Firefly luciferase (Fluc) has been widely used as a bioluminescent monitor. The ATP linear correlation and exogenous luciferin requirement make it useful in most of current imaging systems. However, the utility of this reporter was still limited by the intensity and decay of the luminescent signal, and the active site and structure of enzyme including the relevant substrate channeling region.This study demonstrated a novel construction of bifunctional enzyme system to improve the luminescence generation of firefly luciferase, by bringing in a luciferin-regenerating enzyme (LRE) fusion expressed to the C terminal of luciferase, between which were connected with peptide linker. The fusion protein constructed with typical type of linker, rigid linker (EAAAK) and flexible linker (GGGGS), were analyzed comparing with the unlinked free enzyme. In vivo and in vitro assessment of the bioluminescence intensity and decaying rate to the series of Fluc-LRE enzyme complex were assayed. The fInding demonstrated that the presence of LRE remarkably enhance the generation of luminescence and remained significant stronger signal than that of the control, and the peptide-linked dual enzyme present more stability and continuation on the signal generation and lower decaying rate on signal recession, especially at low dose of Fluc injection. With the advantage of luminescence intensity and reaction period, the peptide mediated fusion expressed LRE may expand the application of Firefly luciferase on bioluminescence imaging.