Increased Substrate Affinity Research Articles

Effect of long-term nitrogen addition on the kinetics of phosphatases in a subtropical forest in southern China

Numerous studies have explored the effects of nitrogen (N) addition on the maximum rate of substrate conversion (Vmax) of phosphatase activity, whereas how N fertilization affects the substrate affinity (the Michaelis constant, Km) of phosphatases is not well understood. Here, we examined a recently-proposed novel hypothesis suggesting that N addition enhances the adaptability of soil microorganisms to low phosphorus (P) conditions by both increasing phosphatase abundance (i.e., increasing the Vmax of phosphatases) and enhancing the substrate-binding affinity of phosphatases (i.e., diminishing the Km of phosphatases). Contrary to the hypothesis, our study found that no level of N addition led to changes in either Vmax or Km of phosphatases in our study site. Furthermore, we observed a positive correlation between Vmax and Km, which challenges the proposed hypothesis, as this positive relationship contradicts the condition suggested by the hypothesis that the adaptive response of increasing phosphatase abundance (higher Vmax) should be associated with increased substrate affinity (lower Km). In conclusion, the present study indicates that N addition does not necessarily enhance the substrate affinity of phosphatases.

Thermostable α-Amylases and Laccases: Paving the Way for Sustainable Industrial Applications

The growing demand in industrial and biotechnological settings for more efficient enzymes with enhanced biochemical features, particularly thermostability and thermotolerance, necessitates a timely response. Renowned for their versatility, thermostable enzymes offer significant promise across a range of applications, including agricultural, medicinal, and biotechnological domains. This comprehensive review summarizes the structural attributes, catalytic mechanisms, and connection between structural configuration and functional activity of two major classes of thermostable enzymes: α-amylases and laccases. These enzymes serve as valuable models for understanding the structural foundation behind the thermostability of proteins. By highlighting the commercial importance of thermostable enzymes and the interest these generate among researchers in further optimization and innovation, this article can greatly contribute to ongoing research on thermostable enzymes and aiding industries in optimizing production processes via immobilization, use of stabilizing additives, chemical modification, protein engineering (directed evolution and mutagenesis), and genetic engineering (through cloning and expression of thermostable genes). It also gives insights to the exploration of suitable strategies and factors for enhancing thermostability like increasing substrate affinity; introducing electrostatic, intramolecular, and intermolecular hydrophobic interactions; mitigating steric hindrance; increasing flexibility of an active site; and N- and C-terminal engineering, thus resulting in heightened multipronged stability and notable enhancements in the enzymes’ industrial applicability.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Structural basis of aggregate binding by the AAA+ disaggregase ClpG

Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a β-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

Heterologous expression and characterization of salt-tolerant β-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-β-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-β-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-β-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45°C, respectively. rBGL_MK86 acted on the β-1,3-linked glucose dimer laminaribiose, but not β-1,4-linked or β-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of β-1,3 glucan) from Laminaria digitata than laminarin (β-1,3/β-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on β-1,6-linked glucose residues. The β-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

Persulfidation maintains cytosolic G6PDs activity through changing tetrameric structure and competing cysteine sulfur oxidation under salt stress in Arabidopsis and tomato.

Glucose-6-phosphate dehydrogenases (G6PDs) are essential regulators of cellular redox. Hydrogen sulfide (H2 S) is a small gasotransmitter that improves plant adaptation to stress; however, its role in regulating G6PD oligomerization to resist oxidative stress remains unknown in plants. Persulfidation of cytosolic G6PDs was analyzed by mass spectrometry (MS). The structural change model of AtG6PD6 homooligomer was built by chemical cross-linking coupled with mass spectrometry (CXMS). We isolated AtG6PD6C159A and SlG6PDCC155A transgenic lines to confirm the invivo function of persulfidated sites with the g6pd5,6 background. Persulfidation occurs at Arabidopsis G6PD6 Cystine (Cys)159 and tomato G6PDC Cys155, leading to alterations of spatial distance between lysine (K)491-K475 from 42.0 Å to 10.3 Å within the G6PD tetramer. The structural alteration occurs in the structural NADP+ binding domain, which governs the stability of G6PD homooligomer. Persulfidation enhances G6PD oligomerization, thereby increasing substrate affinity. Under high salt stress, cytosolic G6PDs activity was inhibited due to oxidative modifications. Persulfidation protects these specific sites and prevents oxidative damage. In summary, H2 S-mediated persulfidation promotes cytosolic G6PD activity by altering homotetrameric structure. The cytosolic G6PD adaptive regulation with two kinds of protein modifications at the atomic and molecular levels is critical for the cellular stress response.

Effect of ultra-violet light radiation on Scenedesmus vacuolatus growth kinetics, metabolic performance, and preliminary biodegradation study.

This study presents the effect of ultra-violet (UV) light radiation on the process kinetics, metabolic performance, and biodegradation capability of Scenedesmus vacuolatus. The impact of the UV radiation on S. vacuolatus morphology, chlorophyll, carotenoid, carbohydrates, proteins, lipid accumulation, growth rate, substrate affinity and substrate versatility were evaluated. Thereafter, a preliminary biodegradative potential of UV-exposed S. vacuolatus on spent coolant waste (SCW) was carried out based on dehydrogenase activity (DHA) and total petroleum hydrocarbon degradation (TPH). Pronounced structural changes were observed in S. vacuolatus exposed to UV radiation for 24h compared to the 2, 4, 6, 12 and 48h UV exposure. Exposure of S. vacuolatus to UV radiation improved cellular chlorophyll (chla = 1.89-fold, chlb = 2.02-fold), carotenoid (1.24-fold), carbohydrates (4.62-fold), proteins (1.44-fold) and lipid accumulations (1.40-fold). In addition, the 24h UV exposed S. vacuolatus showed a significant increase in substrate affinity (1/Ks) (0.959), specific growth rate (µ) (0.024h-1) and biomass accumulation (0.513g/L) by 1.50, 2 and 1.9-fold respectively. Moreover, enhanced DHA (55%) and TPH (100%) degradation efficiency were observed in UV-exposed S. vacuolatus. These findings provided major insights into the use of UV radiation to enhance S. vacuolatus biodegradative performance towards sustainable green environment negating the use of expensive chemicals and other unfriendly environmental practices.

Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters.

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the invitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type ofSBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

Regulation of alanine racemase activity by carboxylates and the d-type substrate d-alanine.

Alanine racemases (ALRs) are essential for d-alanine (d-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in Km , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylate-binding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of d-Ala at high concentrations, implying that d-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and d-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.

The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

DNA-modulated single-atom nanozymes with enhanced enzyme-like activity for ultrasensitive detection of dopamine.

Despite the current progress in optimizing and tailoring the performance of nanozymes through structural and synthetic adaptation, there is still a lack of dynamic modulation approaches to alter their catalytic activity. Here, we demonstrate that DNA can act as an auxiliary regulator via a straightforward incubation method with Fe-N-C single-atom nanozymes (SAzymes), causing a leap in the enzyme-like activity of Fe-N-C from moderate to a higher level. The DNA-assisted enhancement is attributed to the increased substrate affinity of Fe-N-C nanozymes through electrostatic attraction between the substrate and DNA. Based on the prepared DNA/Fe-N-C system, colorimetric sensors for dopamine (DA) detection were constructed. Surprisingly, the incorporation of DNA not only enabled the detection of DA in a low concentration range, but also greatly improved the sensitivity with a 436-fold decrease in detection limit. The quantitative determination of DA was achieved in two-segment linear ranges of 0.01-4 μM and 5-100 μM with an ultralow detection limit of 9.56 nM. The DNA/Fe-N-C system shows superior performance compared to the original Fe-N-C system, making it an ideal choice for nanozyme-based biosensors. This simple design approach has paved the way for enhancing nanozyme activity and is expected to serve as a general strategy for optimizing biosensor performance.

Role of C-terminal domain in a manganese-catalase from Geobacillus thermopakistaniensis

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. We have characterized two manganese-catalases from Geobacillus thermopakistaniensis, CatGt and Cat-IIGt, which exhibited significant variation in their sequence, structure and properties. There was only 23% sequence identity between the two. The striking structural difference was the presence of an extended C-terminal domain in CatGt. Molecular modelling and docking studies revealed that deletion of the C-terminal domain removes non-specific binding, which results in increased substrate affinity. To verify experimentally, a C-terminal truncated version of CatGt, named as CatGt-ΔC, was produced in Escherichia coli and effects of deletion were analyzed. There was no significant difference in optimal pH, optimal temperature and substrate specificity of CatGt and CatGt-ΔC. However, Km value was reduced from 259 to 157mM and CatGt-ΔC exhibited ∼1.5-fold higher catalytic efficiency as compared to CatGt. Furthermore, removal of the C-terminal domain converted the tetrameric nature to monomeric, and reduced the thermostability of the truncated protein. These results demonstrate that C-terminal domain of CatGt might have little role in maintaining enzyme function but provides additional structural stability to the protein, which is a desired property for industrial applications.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ1–42) fibril formation in vitro and suppressed Aβ1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.

An interference-free new xanthine biosensor based on immobilized enzyme-nanogold conjugate on carbon nanotube doped poly(3,4-Ethylenedioxythiophene) composite film

A new design of biosensor based on polymeric nano(bio)composite has been proposed for the selective detection of xanthine to be used in the clinical analysis as well as food quality control. The xanthine oxidoreductase (XOR) gene ofPseudomonas aerogenosastrain CEBP1 wascloned to obtainpurifiedenzyme through affinity chromatography. fMWCNTdoped PEDOTwas electrodeposited on the working electrodeto enhance the sensitivity and selectivity of the biosensor. Bio-synthesized gold nanoparticles conjugated XOR (Au-XOR) was covalently immobilized on the polymeric nanocomposite. The enzymatic activity was enhanced 1.12 times with increased substrate affinity. The surface morphology and structural properties of the polymeric layer were investigated using SEM, FESEM, TEM. Electrochemical characteristics were performed by cyclic voltammetry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy. Xanthine was oxidized (pH 7.0) on the uniquely designed polymeric nano(bio)composite modified electrode at a lower anodic potential of + 0.446 V vs. Ag/AgCl (3 M NaCl)at optimized DPV conditions. The simple, newly designed Au-XOR/fMWCNT-PEDOT/GCE exhibited interference-free reproducibility and stability (∼4 months) with excellent sensitivity of 16.075 µA.µM−1.cm−2for the quantification of xanthine in biological samples such as blood, tissue, urine. The applicability of thebiosensor was validatedby comparing the sensing results for the real biological fluidic solutions with HPLC data (RE = 0.5–3.1%).

Investigation of changes in protein stability and substrate affinity of 3CL-protease of SARS-CoV-2 caused by mutations.

3CLpro of SARS-CoV-2 is one of the enzymes required for the replication process of the virus responsible for the COVID-19 pandemic. In this study, changes in protein stability and substrate affinity caused by mutations were investigated to stir the development of potent inhibitors. Sequence data of samples were obtained from the NCBI Virus database. Mutation analyses were performed with RDP4 and MegaX. 3CLpro tertiary models were created using Robetta. Molecular docking for peptidomimetic substrate and inhibitor ligand was done with Autodock v4.2 and Haddock v2.4. Protein stability analysis was performed using mCSM stability and DynaMut2. Twenty-four missense mutations in 3CLpro were identified in this study. Changes in the 3CLpro structure induced by the mutations Met49Thr, Leu167Ser, and Val202Ala resulted in significant levels of instability (-2.029,-2.612,-2.177 kcal.mol-1, respectively). The lowest interaction energy for substrate was -58.7 kcal.mol-1 and -62.6 kcal.mol-1 in wild-type and mutant, respectively. The lowest docking energy for ligand was -6.19 and -9.52 kcal.mol-1 for wild-type and mutant, respectively. This study reports for the first time that mutations cause increased substrate affinity of 3CLpro from SARS-CoV-2. This research provides important data for the development of potent peptidomimetic inhibitors for the treatment of COVID-19.

Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using β-galactosidase as a model

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using β-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant β-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized β-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant β-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Single-Point Mutation Near Active Center Increases Substrate Affinity of Alginate Lyase AlgL-CD.

Alginate lyases have been widely used for the preparation of bioactive alginate oligosaccharides. An alginate lyase AlgL-CD was rationally designed by introducing alkaline amino acid residues near active center to increase activity. One of its mutants E226K presented much higher activity than wild-type AlgL-CD. Substrate affinity of E226K increased 10 folds as the Km values indicated. The spectra of intrinsic emission fluorescence and circular dichroism of E226K suggested the whole enzyme turned to be more flexible. The 8-anilino-1-naphthalenesulfonate (ANS)-binding assay showed that the hydrophobic active center of E226K was more available to ligand. Molecular dynamic analysis of the enzyme-substrate complex showed that lid loops of the active center in E226K turned to be more opened up, which might contribute to the increase of substrate-binding affinity. Meanwhile, the catalytic residue of E226K was closer to the hydrogen donor C5 atom of the substrate to increase catalysis rate. The final degradation products of alginate by E226K were determined to be identical with that of AlgL-CD. This study provides guidance for improving enzymatic preparation efficiency of bioactive alginate oligosaccharides.

Impact of the size effect on enzymatic electrochemical detection based on metal-organic frameworks

Metal-organic frameworks (MOFs) hold a great promise as immobilization carriers for enzymes and other biomolecules, owing to their enhanced stability, selectivity and controllability. However, enzyme-MOF complexes usually lead to a decrease in the apparent enzyme activity and apparent substrate affinity as a result of the constrained structure of the enzyme and the mass transfer limitation of the substrate, respectively. These results consequently impede the applications of enzyme-MOF complexes in biocatalysis and biosensing. In this study, zeolitic imidazolate framework-8 (ZIF-8) was synthesized to immobilize cytochrome c (Cyt c) via a one-step co-precipitation process under mild conditions. By adjusting the molar ratio of precursors, enzyme-MOF composites with different sizes from 100 nm to 1.3 μm were prepared. The decreased size of the prepared MOFs generated an increase in substrate affinity (with an over 50% decrease in the Michaelis constant Km) and a 6.4-fold improvement in the apparent enzyme activity with a 6.26-fold increase in the enzymatic electrochemical detection sensitivity compared with native Cyt c. The enzyme-MOF composites were coated on a screen-printed electrode for the sensitive and fast detection of H2O2, which is the most common representative of reactive oxygen species in cellular environments, showing the potential for the construction of efficient biosensors with applications in biomedicine.

Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.