Addition Of Cofactors Research Articles

Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30–90 °C) and pH range (6–8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

Biocatalytic Synthesis of l‐Pipecolic Acid by a Lysine Cyclodeaminase: Batch and Flow Reactors

Abstractl‐Pipecolic Acid (l‐PA) is a valuable building block for the synthesis of pharmaceuticals such as anesthetics and immunosuppressants. Thus, more efficient and greener strategies are desired for its production. Herein, we have applied a previously engineered variant of the Lysine Cyclodeaminase from Streptomyces pristinaespiralis (e‐SpLCD) for the bioconversion of l‐Lysine into l‐PA. The reaction can be performed by the free e‐SpLCD reaching full conversion to 50 mM l‐PA. From a biotechnological perspective, the process scale‐up has been trialed in a SpinChem® reactor, albeit with lower conversion yields. To further enhance the biocatalyst stability, we present a detailed study of the e‐SpLCD immobilization on microparticles. This enabled the integration of the immobilized biocatalyst into a packed‐bed reactor for the continuous flow synthesis of l‐PA. The full conversion was achieved in 90 min, maintaining also high operational stability. Remarkably, the addition of exogenous cofactor was not needed for the flow reaction, although the long‐term operational stability was improved by the addition of NAD+.

Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be differentin vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similarenzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer bepresent if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

Efficient production of γ-aminobutyric acid using engineered Escherichia coli whole-cell catalyst

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5′-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

TET-Yeasate: An engineered yeast whole-cell lysate-based approach for high performance tetracycline degradation

The widespread of tetracycline (TC) residues in anthropogenic and natural environments pose an immediate threat to public health. Herein, we established the TET-Yeasate, an approach based on whole-cell lysate of engineered yeast, to mitigate the TC contamination in environment. The TET-Yeasate is defined as the biological matrix of whole cell lysate from engineered yeast that containing TC-degradative components (Tet(X), NADPH, Mg2+) and protective macromolecules. The TET-Yeasate was able to efficiently eliminate TC residues in tap water (98.8%), lake water (77.6%), livestock sewage (87.3%) and pharmaceutical wastewater (35.3%) without necessity for exogenous addition of expensive cofactors. The TET-Yeasate was further developed into lyophilized form for ease of storage and delivery. The TET-Yeasate in lyophilized form efficiently removed up to 74.6% TC residue within 0.25 h. In addition, the lyophilization confers promising resilience to TET-Yeasate against adverse temperatures and pH by maintaining degradation efficacy of 85.69%−97.83%. The stability test demonstrated that the biomacromolecules in lysate served as natural protectants that exerted extensive protection on TET-Yeasate during the 14-day storage at various conditions. In addition, 5 potential degradation pathways were elaborated based on the intermediate products. Finally, the analysis indicated that TET-Yeasate enjoyed desirable bio- and eco-safety without introduction of hazardous intermediates and spread of resistance genes. To summary, the TET-Yeasate based on whole cell lysate of engineered yeast provides a cost-effective and safe alternative to efficiently remove TC residues in environment, highlighting the great potential of such whole-cell based methods in environmental decontamination.

Glycosylation of polyphenolic compounds: Design of a self-sufficient biocatalyst by co-immobilization of a glycosyltransferase, a sucrose synthase and the cofactor UDP

Glycosyltransferases catalyze the regioselective glycosylation of polyphenolic compounds, increasing their solubility without altering their antioxidant properties. Leloir-type glycosyltransferases require UDP-glucose as a cofactor to glycosylate a hydroxyl of the polyphenol, which is expensive and unstable. To simplify these processes for industrial implementation, the preparation of self-sufficient heterogeneous biocatalysts is needed. In this study, a glycosyltransferase and a sucrose synthase (as an UDP-regenerating enzyme) were co-immobilized onto porous agarose-based supports coated with polycationic polymers: polyethylenimine and polyallylamine. In addition, the UDP cofactor was strongly ionically adsorbed and co-immobilized with the enzymes, eliminating the need to add it separately. Thus, the optimal self-sufficient heterogeneous biocatalyst was able to catalyze the glycosylation of three polyphenolic compounds (piceid, phloretin and quercetin) with in situ regeneration of the UDP-glucose, allowing multiple consecutive reaction cycles without the addition of exogenous cofactor. A TTN value of 50 (theoretical maximum) was obtained in the reaction of piceid glycosylation, after 5 reaction cycles, using the self-sufficient biocatalyst based on an improved sucrose synthase variant. This result was 5-fold higher than the obtained using soluble cofactor and the co-immobilized enzymes, and much higher than those reported in the literature for similar processes.

Fully co-factor-free ClearTau platform produces seeding-competent Tau fibrils for reconstructing pathological Tau aggregates

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients’ brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method – ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.

Localization of multiple DNAzymes as a branchedzyme-powered nanodevice for the immunoassay of tumor biomarkers

Traditional immunoassay methods often face challenges due to the labeling procedure of protein enzymes, the use of multiple antibodies, and severe conditions. To address these limitations, we propose the concept of incorporating branchedzyme-powered nanodevices into immunoassays. In this strategy, multiple DNAzymes are localized onto gold nanoparticles (AuNPs) along with substrates. The localization format facilitates intramolecular reactions between DNAzymes and substrates, leading to accelerated kinetics of the nanodevice. Upon the formation of an immunocomplex with an antibody on a 96-well plate, the branchedzyme-powered nanodevice catalytically releases multiple fluorescent signals under ambient temperature, eliminating the need for secondary antibodies. The branched DNAzymes exhibit catalytic properties similar to those of protein enzymes, thus simplifying the assay procedure and achieving isothermal detection. Furthermore, the detection process can be controlled by the addition or deletion of cofactors. Additionally, the affinity ligand can be easily modified to construct nanodevices specific to different targets without requiring extensive redesign. This strategy has demonstrated successful quantification of tumor biomarkers such as alpha-fetoprotein (AFP) and prostate-specific antigen (PSA) at subpicomolar concentrations, showcasing its suitability for clinical applications. Consequently, the branchedzyme-powered nanodevice represents a valuable addition to the immunoassay toolbox, opening new possibilities for clinical diagnostics.

Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis.

Lacto-N-biose (LNB) is a member of the human milk oligosaccharide (HMO) family and is synthesized via an enzymatic reaction in vitro with N-acetylglucosamine (GlcNAc) and cofactors. In this study, LNB was synthesized using a cell factory for the first time. First, three modules were constructed in Kluyveromyces lactis for producing LNB from lactose and GlcNAc without the addition of cofactors. Second, a de novo pathway was constructed in K. lactis for producing LNB from lactose without adding GlcNAc. Finally, a transcriptional switch was introduced into K. lactis to reprogram its metabolic network for improving the flux from GlcNAc-6-P to GlcNAc in the de novo pathway. Subsequently, a final LNB yield of 10.41 g/L, similar to the salvage pathway yield, was achieved through the de novo pathway. The engineered K. lactis provides a promising technology platform for the industrial scale production of LNB.

Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli.

γ-Aminobutyric acid (GABA) is used widely in various fields, such as agriculture, food, pharmaceuticals, and biobased chemicals. Based on glutamate decarboxylase (GadBM4) derived from our previous work, three mutants, GadM4-2, GadM4-8, and GadM4-31, were obtained by integrating enzyme evolution and high-throughput screening methods. The GABA productivity obtained through whole-cell bioconversion using recombinant Escherichia coli cells harboring mutant GadBM4-2 was enhanced by 20.27% compared to that of the original GadBM4. Further introduction of the central regulator GadE of the acid resistance system and the enzymes from the deoxyxylulose-5-phosphate-independent pyridoxal 5'-phosphate biosynthesis pathway resulted in a 24.92% improvement in GABA productivity, reaching 76.70 g/L/h without any cofactor addition with a greater than 99% conversion ratio. Finally, when one-step bioconversion was applied for the whole-cell catalysis in a 5 L bioreactor, the titer of GABA reached 307.5 ± 5.94 g/L with a productivity of 61.49 g/L/h by using crude l-glutamic acid (l-Glu) as the substrate. Thus, the biocatalyst constructed above combined with the whole-cell bioconversion method represents an effective approach for industrial GABA production.

Hand-in-hand structured DNA monolayer for dual-mode analysis of circulating tumor DNA

DNA nanostructures with different functions have been extensively studied and applied. Controllable and programmable assembly of functional DNA nanostructures still requires in-depth investigation to guarantee accurate and reliable practical utility. In this study, we have extended the conception of common molecular beacon for the fabrication of novel DNA nanostructures. Three hairpin fuel strands are designed for target circulating tumor DNA triggered construction of three-way junction structures. By the conjugation of swing arms, hand-in-hand DNA monolayer is achieved at the electrode surface. On one hand, electrochemical species could be located near electrode interface due to this structure for electrochemical output; on the other, the hand-in-hand pattern could be cleaved after the addition of DNAzyme cofactor and the released strands carry abundant carbon nanodots for fluorescent out. With the dual-mode analysis, highly selective and reliable detection of target is achieved. The high sensitivity of the proposed method is attributed to the recycling of target strands during the formation of multiple three-way junctions. It holds great potential for the application of DNA analysis and clinical diagnosis.

Evaluation and simplification of risk factors in FIGO 2000 scoring system for gestational trophoblastic neoplasia: a 19-year retrospective analysis.

The International Federation of Gynecology and Obstetrics (FIGO) 2000 scoring system classifies gestational trophoblastic neoplasia (GTN) patients into low- and high-risk groups, so that single- or multi-agent chemotherapy can be administered accordingly. However, a number of FIGO-defined low-risk patients still exhibit resistance to single-agent regimens, and the risk factors currently adopted in the FIGO scoring system possess inequable values for predicting single-agent chemoresistance. The purpose of this study is therefore to evaluate the efficacy of risk factors in predicting single-agent chemoresistance and explore the feasibility of simplifying the FIGO 2000 scoring system for GTN. The clinical data of 578 GTN patients who received chemotherapy between January 2000 and December 2018 were retrospectively reviewed. Univariate and multivariate logistic regression analyses were carried out to identify risk factors associated with single-agent chemoresistance in low-risk GTN patients. Then, simplified models were built and compared with the original FIGO 2000 scoring system. Among the eight FIGO risk factors, the univariate and multivariate analyses identified that pretreatment serum human chorionic gonadotropin (hCG) level and interval from antecedent pregnancy were consistently independent predictors for both first-line and subsequent single-agent chemoresistance. The simplified model with two independent factors showed a better performance in predicting single-agent chemoresistance than the model with the other four non-independent factors. However, the addition of other co-factors did improve the efficiency. Overall, simplified models can achieve favorable performance, but the original FIGO 2000 prognostic system still features the highest discrimination. Pretreatment serum hCG level and interval from antecedent pregnancy were independent predictors for both first-line and subsequent single-agent chemoresistance, and they had greater weight than other non-independent factors in predicting single-agent chemoresistance. The simplified model composed of certain selected factors is a promising alternative to the original FIGO 2000 prognostic system, and it shows comparable performance.

Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts.

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis–Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

Application of dual carbon-bromine stable isotope analysis to characterize anaerobic micro-degradation mechanisms of PBDEs in wetland bottom-water

Polybrominated diphenyl ethers (PBDEs), one kind of persistent organic pollutants, were widely detected in coastal wetlands. Microbial reductive debromination is one of the most important attenuation processes for PBDEs in anaerobic environment, whereas the underlying reaction mechanisms remain elusive. Dual-element stable isotope analysis was recently recognized to distinguish different reaction mechanism for degradation of organic pollutants. In this study, the dual carbon-bromine isotope effects associated with the anaerobic microbial degradation were first investigated to characterize the reaction mechanisms for BDE-47 and BDE-153. Presence of lower brominated congeners indicated stepwise debromination as the main degradation pathway, with the preferential removal of bromine in para position > meta/ortho position. The pronounced isotope fractionation was observed for both carbon and bromine, with similar carbon (εC) and bromine isotope enrichment factor (εBr) between BDE-47 (εC = -5.98‰, εBr = -2.44‰) and BDE-153 (εC = -5.57‰, εBr = -2.06‰) during the microbial degradation. Compared to εC and εBr, the correlation of carbon and isotope effects (ΛC/Br = Δδ81Br/Δδ13C) was almost the same between BDE-47 (0.436) and BDE-153 (0.435), indicating the similar reaction mechanism. The calculated carbon and bromine apparent kinetic isotope effects (AKIEC and AKIEBr) were 1.0773 and 1.0098 for BDE-47 and 1.0716 and 1.0125 for BDE-153, within range reported for degradation of halogenated compounds following nucleophilic substitution. Combination analysis of degradation products, ΛC/Br and AKIE, all the results pointed to that the anaerobic reductive debromination of BDE-47 and BDE-153 followed the nucleophilic aromatic substitution, with the addition of cofactor to the benzene ring concomitant with dissociation of carbon-bromine bond via the inner-sphere electron transfer, and the cleavage of C-Br bond was the rate-determining step. This study contributed to the development of dual carbon-bromine isotope analysis as a robust approach to probe the fate of PBDEs in contaminated sites.

A novel and robust 3-quinuclidinone reductase from Kaistia algarum for efficient synthesis of (R)-3-quinuclidinol without external cofactor

A novel ketone reductase KaKR was identified from Kaistia algarum through genome mining with high activity and enantioselectivity toward 3-quinuclidione. Sequence alignment and conserved motifs analysis revealed that KaKR was classified into classical short-chain dehydrogenase/reductase family. KaKR displayed the highest activity at pH 7.0 and 50°C. Substrate spectrum analysis revealed that KaKR exhibited high substrate specificity toward N-heterocyclic ketones. The kcat/KM toward 3-quinuclidione and NADH were 82.8 and 1697 s–1⋅mM–1. KaKR is a robust biocatalyst and could tolerate high concentrations of 3-quinuclidione. Without assistance of external cofactor, as high as 5.0 M 3-quinuclidione could be completely and enantioselectivity reduced into (R)-quinuclidinol with ee of >99.9%, S/C of 42.6 g⋅g–1 and space-time yield of 1027 g⋅L–1⋅d–1. Alanine scanning was performed to elucidate the molecular mechanism of the high substrate specificity and enantioselectivity. Two critical residues, V94 and E198, were identified with important roles in determining the catalytic efficiency and enantioselectivity. This study provides a novel and robust ketone reductase, and establishes an efficient and economic bioprocess for the synthesis of (R)-3-quinuclidinol without addition of external cofactor.

The Effect of Addition Mn2+ Metal Ions and Incubation Time to Bacillus cereus Cellulase Enzyme Activity from Endophytic Bacteria of Curcuma Rhizome (Curcuma zanthorrizha Roxb.)

Cellulase is one of the most widely used enzymes in the industrial world and wastes decomposition process. Bacillus cereus is one of the bacteria that can produce cellulase enzymes that can hydrolyze cellulose to glucose. The addition of cofactors and incubation time can help determine the optimum conditions needed by cellulase enzymes to work optimally. This study aims to determine the effect of adding Mn2+ metal ions and incubation time to the activity of cellulase enzymes from Bacillus cereus endophytic bacteria. This research is experimentally used a Completely Randomized Design (CRD) factorial design with two factor treatments and 3 times repetitions. The first factor is variation of addition Mn2+ metal ions which are 5 mM, 10 mM, and 15 mM, the second is variation of incubation time which are 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours. The data were analyzed using Analysis Of Variance (ANOVA) and if the data significantly affected the parameter, then it would be continued by Duncan Multiple Range Test (DMRT) with the fault level of 5%. The result showed that the interaction of addition Mn2+ metal ions and incubation time affected the cellulase enzyme activity of Bacillus cereus from endophytic bacteria. The highest cellulase enzyme activity obtained from interaction addition Mn2+ metal 10 mM and incubation time 3 hours with an activity 0,335 U/mL, while the lowest cellulase enzyme activity obtained from interaction addition Mn2+ metal 15 mM and incubation time 5 hours with an activity of 0,073 U/mL

Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration

SummaryHuman milk oligosaccharides (HMOs), the third most abundant solid component of human milk, are reported to be beneficial to infant health. The biosynthesis of lacto-N-biose (LNB), the building block for HMOs, suffers from excessive addition of cofactors and intermediate inhibition. Here, we developed an in vitro multienzyme cascade composed of LNB module, ATP regeneration, and pyruvate oxidase-driven phosphate recycling to produce LNB. The integration between ATP regeneration and Pi alleviation increased the LNB conversion ratio and resulted in a ΔG'° decrease of 540 KJ/mol. Under optimal conditions, the LNB conversion ratio was improved from 0.34 to 0.83 mol/mol GlcNAc and the ATP addition decreased to 50%. Finally, 0.96 mol/mol GlcNAc and 71.6 mg LNB g−1 GlcNAc h−1 of LNB yield was achieved in a 100-mL reaction system. The synergistic strategy not only paves the way for producing LNB but also facilitates other chemicals with multienzyme cascades.

Structural and Molecular Dynamics of Mycobacterium tuberculosis Malic Enzyme, a Potential Anti-TB Drug Target.

Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ contributes to lipid biosynthesis by providing reductants in the form of NAD(P)H. Here, we present the X-ray crystal structure of MEZ to 3.6 Å. We use biochemical assays to demonstrate MEZ is dimeric in solution and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually flexible, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that dimeric MEZ subunits alternate between open and closed states, and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb and not human malic enzyme homologues.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.