Sequential Enzymatic Reactions Research Articles

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.

Oxindole–curcumin hybrid compound enhances the transcription of γ-glutamylcysteine ligase

Glutathione (GSH), which is particularly important for antioxidant defenses, is synthesized in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine ligase (GCL) and GSH synthase. GCL comprises catalytic (GCLC) and regulatory subunits and catalyzes the rate-limiting step in de novo GSH synthesis. Accumulating evidence suggests that substances that stimulate GSH synthesis are therapeutic modalities for neurodegenerative disorders and schizophrenia, in which a deficit in brain GSH content has been observed. In the present study, we attempted to develop small organic compounds that increase GCLC transcription. Using HT22 cells stably expressing a luciferase reporter that contains rat GCLC promoter region (−1764 to +2), we assessed the effects of the novel neuroprotective compound oxindole and related compounds on GCLC promoter activity. Among approximately 220 synthesized compounds, five compounds increased GCLC promoter activity by >200% at a concentration of 50 μM, and 16 compounds increased promoter activity by approximately 150%. The most effective compound oxindole–curcumin hybrid GIF-2165X-G1 increased GCLC mRNA levels in HT22 mouse hippocampal cells, PC12 rat pheochromocytoma cells, and C6 rat glioma cells. Although GIF-2165X-G1 potently induced antioxidant response element (ARE)-driven transcription, the compound increased GCLC transcriptional activity through Sp1 pathway in a Keap1–Nrf2–ARE-independent manner. These results suggest that GIF-2165X-G1 itself and further modification of the compound are useful interventions for promoting neuronal survival by augmenting resistance to oxidative stress.

Dynamic behavior of liquid droplets with enzyme compartmentalization triggered by sequential glycolytic enzyme reactions.

Dynamic droplet formation via liquid-liquid phase separation (LLPS) is believed to be involved in the regulation of various biological processes. Here, a model LLPS system coupled with a sequential glycolytic enzymatic reaction was developed to reproduce the dynamic control of liquid droplets; (i) the droplets, which consist of poly-L-lysine and nucleotides, compartmentalize two different enzymes (hexokinase and glucose-6-phosphate dehydrogenase) individually, accelerating the overall reaction, and (ii) each enzymatic reaction triggers the formation, dissolution and long-term retention of the droplets by converting the scaffold nucleotides. This model system will offer a new aspect of enzymes associated with LLPS in living cells.

Neddylation blockade induces HIF-1\u03b1 driven cancer cell migration via upregulation of ZEB1

Neddylation is a process by which NEDD8 is covalently conjugated to target proteins by sequential enzymatic reaction. Its role in cancer cell migration has only been recently acknowledged. Previously in cancer cell migration, the epithelial to mesenchymal transition (EMT) process has been well-known to play an important role in both invasion and metastasis by promoting mesenchymal phenotype in epithelial cells. However, the role of neddylation in the EMT process and its mechanistic details are yet to be elucidated. We recently reported that neddylation plays a crucial role in cancer cell migration through the PI3K-Akt pathway. Here, we report that inhibiting neddylation activates the hypoxia-inducible factor 1α (HIF-1α) through the PI3K-Akt pathway, which eventually regulates the EMT-activator ZEB1 (zinc finger E-box binding homeobox 1) in various cancer cell lines. As induction of HIF-1α is known to deteriorate the state of cancer and EMT process is one of the hallmarks of metastasis in cancer, our findings uncover the role of neddylation between HIF-1α and ZEB1.

Abstract 4873: Molecular mechanism of neddylation-dependent suppression of cancer cell migration

Abstract Neddylation is a process by which NEDD8 is covalently conjugated to target proteins by sequential enzymatic reaction. Its role in cancer cell migration has only been recently acknowledged. Previously in cancer cell migration, the epithelial to mesenchymal transition (EMT) process has been well-known to play an important role in both invasion and metastasis by promoting mesenchymal phenotype in epithelial cells. However, the role of neddylation in the EMT process and its mechanistic details are yet to be elucidated. We recently reported that neddylation plays a crucial role in cancer cell migration through the PI3K-Akt pathway. Here, we report that inhibiting neddylation activates the hypoxia-inducible factor-1α through the PI3K-Akt pathway, which eventually regulates the EMT-activator zinc finger E-box binding homeobox 1 in various cancer cell lines. As induction of hypoxia-inducible factor-1α is known to deteriorate the state of cancer and EMT process is one of the hallmarks of metastasis in cancer, our findings uncover the novel association of hypoxia-inducible factor-1α and zinc finger E-box binding homeobox 1 through the process of neddylation. Citation Format: Yang-Sook Chun, Jieun Seo, Jun Bum Park. Molecular mechanism of neddylation-dependent suppression of cancer cell migration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4873.

ATP-dependent hydroxylation of an unactivated primary carbon with water

Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C–H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C–H activations beyond canonical oxygenase-dependent reactions.

Biophysical effects of polystyrene nanoparticles on Elliptio complanata mussels.

The presence of nanoplastics (NPs) in various products and from the weathering of released plastic materials are of concern for the environment's safety. The purpose of this study was to examine the biophysical effects of polystyrene NPs on freshwater mussels. Mussels were exposed to a range of concentrations of NPs (0.1, 0.5, 1, and 5mg/L) for 24 h and allowed to depurate for 12 h in clean aquarium water. The digestive gland was isolated and analyzed for NPs, lipids, viscosity, protein aggregation, anisotropic changes (liquid crystals: LCs), and the oscillatory modulation in viscosity during the formation of self-organizing enzyme complex of fumarase, malate dehydrogenase, and citrate synthase. The results revealed that mussels accumulated NPs in the digestive gland and their levels were significantly correlated with lipids levels, LCs, the increase in the malate dehydrogenase/citrate synthase activity ratio, and oscillations in viscosity. Protein aggregation was also found to be correlated with lipid levels. The data suggests that the presence of NPs in the digestive gland involves changes in lipid content and LC formation and perturbs the normal oscillations in viscosity during sequential enzyme reactions of the above enzymes. It is concluded that the uptake of NPs in cells could disrupt the internal organization of cells which can interfere with the normal association of enzymes involved in energy metabolism.

Acetylcholinesterase-choline oxidase-based mini-reactors coupled with silver solid amalgam electrode for amperometric detection of acetylcholine in flow injection analysis

A stable and simple amperometric biosensor based on the separated bienzyme (acetylcholinesterase (AChE) – choline oxidase (ChOx)) part has been successfully developed for the determination of acetylcholine (ACh) using flow injection analysis for the first time. The bienzyme part consists of two consecutively connected enzymatic mini-reactors filled by mesoporous silica powders (SBA−15) covalently covered by AChE and ChOx, respectively. The oxygen consumption as the result of two sequential enzymatic reactions was monitored amperometrically. The wall-jet cell with working silver solid amalgam electrode covered by mercury film was used for four-electron oxygen reduction at the highly negative potential. The experimental parameters including the amounts of immobilized enzymes, pH of the carrier solution, the detection potential, flow rate, and injection volume of ACh were optimized. The detection limit was found to be 4.1 μmol L−1. The interfering effect of 1.0 mmol L−1 glucose, 0.1 mmol L−1 uric acid, and 0.1 mmol L−1 ascorbic acid to 0.1 mmol L−1 ACh was 0.6%, 2.2%, and 2.8%, respectively. The developed amperometric ACh biosensor showed 59.8% of its initial response after >400 measurements over 100 days.

Construction of artificial membrane transport metabolons - an emerging strategy in metabolic engineering.

The term 'membrane transport metabolon' refers to the physical association of membrane transporters with enzymes that metabolize the transported substrates. In naturally evolved systems, physiological relevance of coupling transport with sequential enzymatic reactions resides, for instance, in faster turnover rates, protection of substrates from competing pathways or shielding the cellular environment from toxic compounds. Such underlying principles offer attractive possibilities for metabolic engineering approaches and concepts for constructing artificial transporter-enzyme complexes are recently being developed. In this minireview, the modes of substrate channeling across biological membranes and design principles for artificial transport metabolons are discussed.

Fluorescence detection of metabolic activity of the fatty acid beta oxidation pathway in living cells.

Detection of metabolic activity in living cells facilitates the understanding of the cell mechanism. Here, we report a fluorescent probe that can detect fatty acid beta oxidation (FAO) in living cells. This probe is metabolically degraded by the sequential enzyme reactions of FAO and can visualize the FAO activity with turn-on fluorescence.

The Molecular Mechanisms Underlying Iron Deficiency Responses in Rice.

Iron (Fe) is an essential element required for plant growth and development. Under Fe-deficientconditions, plants have developed two distinct strategies (designated as strategy I and II) to acquire Fe from soil. As a graminaceous species, rice is not a typical strategy II plant, as it not only synthesizes DMA (2′-deoxymugineic acid) in roots to chelate Fe3+ but also acquires Fe2+ through transporters OsIRT1 and OsIRT2. During the synthesis of DMA in rice, there are three sequential enzymatic reactions catalyzed by enzymes NAS (nicotianamine synthase), NAAT (nicotianamine aminotransferase), and DMAS (deoxymugineic acid synthase). Many transporters required for Fe uptake from the rhizosphere and internal translocation have also been identified in rice. In addition, the signaling networks composed of various transcription factors (such as IDEF1, IDEF2, and members of the bHLH (basic helix-loop-helix) family), phytohormones, and signaling molecules are demonstrated to regulate Fe uptake and translocation. This knowledge greatly contributes to our understanding of the molecular mechanisms underlying iron deficiency responses in rice.

Controlled Release of a Micelle Payload via Sequential Enzymatic and Bioorthogonal Reactions in Living Systems

AbstractA bioorthogonal approach is explored to release the content of nanoparticles on demand. Exploiting our recently described click‐and‐release technology, we developed a new generation of cleavable micelles able to disassemble through a sequential enzymatic and bioorthogonal activation process. Proof‐of‐concept experiments showed that this new approach could be successfully used to deliver the substances encapsulated into micelles in living cells as well as in mice by two complementary targeted strategies.

Controlled Release of a Micelle Payload via Sequential Enzymatic and Bioorthogonal Reactions in Living Systems.

A bioorthogonal approach is explored to release the content of nanoparticles on demand. Exploiting our recently described click-and-release technology, we developed a new generation of cleavable micelles able to disassemble through a sequential enzymatic and bioorthogonal activation process. Proof-of-concept experiments showed that this new approach could be successfully used to deliver the substances encapsulated into micelles in living cells as well as in mice by two complementary targeted strategies.

MODELING OF THE BIOSYNTHESIS OF LIGNIN AS A PROCESS OF ENZYMATIC DEHYDROPOLYMERIZATION MONOLIGNOLS

The results of theoretical modeling of lignin biosynthesis in the system of peroxidase-hydrogen peroxide-monolignol are presented. The process under study is presented as a sequence of enzymatic and chemical reactions occurring in an open system. The growth of the lignin molecule is due to successive stages of the formation of phenoxyl radicals of different generation numbers and acts of recombination of these radicals. The mathematical model of the process in the form of a system of 14 differential equations is proposed for the first time. For the numerical solution of the system, the eighth order Runge-Kutta-Fehlberg method with an automatic integration step was applied. The results of numerical integration are presented, which indicate a significant dependence of the process dynamics on the initial conditions. Various modes of the process, including oscillatory modes, are found. Features of the system dynamics are due to the choice of the initial concentration of monolignol, hydrogen peroxide and the enzyme, as well as the nonlinearity of differential equations and the presence of feedbacks. The results of numerical experiments within the framework of the first proposed mathematical concept on dehydropolymerization of monolignols allow us to give a theoretical justification of the phenomena that are currently treated as the processes of dynamic self-organization in the biosynthesis of this polymer.

Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

Stepwise Synthesis of Quercetin Bisglycosides Using Engineered Escherichia coli.

Synthesis of flavonoid glycoside is difficult due to diverse hydroxy groups in flavonoids and sugars. As such, enzymatic synthesis or biotransformation is an approach to solve this problem. In this report, we used stepwise biotransformation to synthesize two quercetin bisglycosides (quercetin 3-O-glucuronic acid 7-O-rhamnoside [Q-GR] and quercetin 3-O-arabinose-7-O-rhamnoside [Q-AR]) because quercetin O-rhamnosides contain antiviral activity. Two sequential enzymatic reactions were required to synthesize these flavonoid glycosides. We first synthesized quercetin 3-O-glucuronic acid [Q-G], and quercetin 3-O-arabinose-[Q-A] from quercetin using E. coli harboring specific uridine diphopsphate glycosyltransferase (UGT) and genes for UDP-glucuronic acid and UDP-arabinose, respectively. With each quercetin 3-O-glycoside, rhamnosylation using E. coli harboring UGT and the gene for UDP-rhamnose was conducted. This approach resulted in the production of 44.8 mg/l Q-GR and 45.1 mg/l Q-AR. This stepwise synthesis could be applicable to synthesize various natural product derivatives in case that the final yield of product was low due to the multistep reaction in one cell or when sequential synthesis is necessary in order to reduce the synthesis of byproducts.

Paper-based luminescence bioassay method embedding a sequence of enzymatic reactions to detect sulfonamide groups

Sulfonamide residue in foodstuffs and the environment is a serious global concern for their contribution to the occurrence of antibiotic-resistant bacteria, especially in developing countries. Here, we describe a novel, simple, and low-cost bioassay for sulfonamides, which has high potential versatility for use in low-resource settings. The bioassay method is based on a purpose-built luminescent assay reaction that detects sulfonamide groups. The luminescent assay reaction comprises dihydropteroate synthase, a target enzyme of sulfonamides, and luminescent pyrophosphate detection reagent, which triggers a sequence of biomolecular reactions that convert sulfonamides to emit luminescence. The novel assay detected at least six different sulfonamides with an estimated limit of detection of <25 ng ml-1 in a solution-phase assay using a microplate reader. More importantly, the luminescent assay reaction functioned even after spotting and freeze-drying on a wax pattern-printed paper platform. The paper-embedded luminescent assay reaction showed response signals to sulfadiazine within 30 min at a limit of detection similar to that of the solution-phase assay using a microplate reader. The signal could be recorded using a digital camera in the dark and required no other laboratory infrastructure, freeing the assay from the constraints of a well-fitted laboratory.

Foldable paper-based analytical device for the detection of an acetylcholinesterase inhibitor using an angle-based readout

The rapid on-site detection of organophosphate compounds such as nerve agents and pesticides is essential due to their lethal effect, which irreversibly inhibits the activity of acetylcholinesterase (AChE). In this study, we demonstrate a foldable paper-based analytical device (PAD) that is capable of detecting an AChE inhibitor in a semiquantitative manner. An angle-based readout of the yellow area in the ring-type paper channel was used for the semiquantitative analysis with the naked eye. The color of the paper channel was changed to yellow as a result of oxidized cerium oxide nanoparticles by hydrogen peroxide produced from the enzymatic reactions. All reagents required for enzyme reactions were preadsorbed on the pop-up card shaped PAD to minimize the manual steps for dealing with multiple reagents for sequential enzyme reactions. As a proof of concept, we were able to semiquantitatively analyze a nerve agent simulant called dimethyl methylphosphonate (DMMP), one of the AChE inhibitors, by simply reading the angle of the yellow area in the paper channel. The foldable PAD proposed here would serve as a valuable tool that allows unskilled people to semiquantitatively detect AChE inhibitors in the field, including nerve agents and organophosphate pesticides.

Flow system turns amines into alcohols efficiently

A new closed-loop flow reaction system improves the yield and speed of enzymatic reaction sequences that convert amines—including dopamine and other biogenic amines—to alcohols with the same chiral...

Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S.

The denitrifying betaproteobacterium Sterolibacterium denitrificans Chol1S catabolizes steroids such as cholesterol via an oxygen-independent pathway. It involves enzyme reaction sequences described for aerobic cholesterol and bile acid degradation as well as enzymes uniquely found in anaerobic steroid-degrading bacteria. Recent studies provided evidence that in S. denitrificans, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ4-dafachronic acid (C26-oic acid), which is subsequently activated by a substrate-specific acyl-coenzyme A (acyl-CoA) synthetase (ACS). Further degradation was suggested to proceed via unconventional β-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical β-hydroxyacyl-CoA dehydrogenases and thiolases. Here, we heterologously expressed three cholesterol-induced genes that putatively code for AMP-forming ACSs and characterized two of the products as specific 3β-hydroxy-Δ5-cholenoyl-CoA (C24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C26-, C24-, or C22-oic-acids but activated 3aα-H-4α-(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) to HIP-CoA, a rather late intermediate of aerobic cholesterol degradation that still contains the CD rings of the sterane skeleton. This work provides experimental evidence that anaerobic steroid degradation proceeds via numerous alternate CoA-ester-dependent or -independent enzymatic reaction sequences as a result of aldolytic side chain and hydrolytic sterane ring C-C bond cleavages. The aldolytic side chain degradation pathway comprising highly exergonic ACSs and aldehyde dehydrogenases is considered to be essential for driving the unfavorable oxygen-independent C26 hydroxylation forward.IMPORTANCE The biological degradation of ubiquitously abundant steroids is hampered by their low solubility and the presence of two quaternary carbon atoms. The degradation of cholesterol by aerobic Actinobacteria has been studied in detail for more than 30 years and involves a number of oxygenase-dependent reactions. In contrast, much less is known about the oxygen-independent degradation of steroids in denitrifying bacteria. In the cholesterol-degrading anaerobic model organism Sterolibacterium denitrificans Chol1S, initial evidence has been obtained that steroid degradation proceeds via numerous alternate coenzyme A (CoA)-ester-dependent/independent reaction sequences. Here, we describe the heterologous expression of three highly specific and characteristic acyl-CoA synthetases, two of which play key roles in the degradation of the side chain, whereas a third one is specifically involved in the B ring degradation. The results obtained shed light into oxygen-independent steroid degradation comprising more than 40 enzymatic reactions.