Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Boosted activity by engineering the enzyme microenvironment in cascade reaction: A molecular understanding.

ABSTRACT: Engineering of enzyme microenvironment can surprisingly boost the apparent activity. However, the underlying regulation mechanism is not well-studied at a molecular level so far. Here, we present a modulation of two model enzymes of cytochrome c (Cty C) and _d-amino acid oxidase (DAAO) with opposite pH-activity profiles using ionic polymers. The operational pH of poly (acrylic acid) modified Cyt C and polyallylamine modified DAAO was extended to 3-7 and 2-10 where the enzyme activity was larger than that at their optimum pH of 4.5 and 8.5 by 106% and 28%, respectively. The cascade reaction catalyzed by two modified enzymes reveals a 1.37-fold enhancement in catalytic efficiency compared with their native counterparts. The enzyme activity boosting is understood by performing the UV-vis/CD spectroscopy and molecular dynamics simulations in the atomistic level. The increased activity is ascribed to the favorable microenvironment in support of preserving enzyme native structures nearby cofactor under external perturbations.

SUBMITTER: Wang J

PROVIDER: S-EPMC8271104 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Forward design of a complex enzyme cascade reaction.

Project description:Enzymatic reaction networks are unique in that one can operate a large number of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biological processes such as glycolysis. For this, we adapt the characterization of the biochemical system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply standard system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chemical production purposes.

| S-EPMC5052792 | biostudies-literature

Biomimetic enzyme cascade reaction system in microfluidic electrospray microcapsules.

Project description:Mimicking subcellular compartments containing enzymes in organisms is considered a promising approach to substitute for missing or lost cellular functions. Inspired by the multicompartment structures of cellular architectures, we present a novel multienzyme system based on hollow hydrogel microcapsules with flexible enzymatic inverse opal particles. Benefiting from the precise operation capability of the microfluidic electrospray and the remarkable structural color marks in the inverse opal particles, we developed a multienzyme system with controllable number, type, and spatial arrangement of the encapsulated enzymes. The hydrogel shells also could improve enzyme stability against proteolysis in the system. The multienzyme system containing alcohol oxidase and catalase could act as a cascade biocatalyst and reduce alcohol levels in media, providing an alternative antidote and prophylactic for alcohol intoxication. These features indicated that our strategy provides an ideal enzyme cascade reaction system for complex biocatalysis and biomimetic functions of some organelles or organs.

| S-EPMC6003728 | biostudies-literature

Understanding bistability in complex enzyme-driven reaction networks.

Project description:Much attention has been paid recently to bistability and switch-like behavior that might be resident in important biochemical reaction networks. There is, in fact, a great deal of subtlety in the relationship between the structure of a reaction network and its capacity to engender bistability. In common physicochemical settings, large classes of extremely complex networks, taken with mass action kinetics, cannot give rise to bistability no matter what values the rate constants take. On the other hand, bistable behavior can be induced in those same settings by certain very simple and classical mass action mechanisms for enzyme catalysis of a single overall reaction. We present a theorem that distinguishes between those mass action networks that might support bistable behavior and those that cannot. Moreover, we indicate how switch-like behavior results from a well-studied mechanism for the action of human dihydrofolate reductase, an important anti-cancer target.

| S-EPMC1592242 | biostudies-literature

A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes.

Project description:Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) - with hydrogen peroxide (H2O2) as HRP "activator" - was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) to 2',7'-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH2-DA/H2O2 substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).

| S-EPMC9053938 | biostudies-literature

A Multi-Enzyme Cascade Reaction for the Production of 2'3'-cGAMP.

Project description:Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

| S-EPMC8073963 | biostudies-literature

Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.

Project description:Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).

| S-EPMC3838371 | biostudies-literature

Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reaction.

Project description:The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in nature.

| S-EPMC7612480 | biostudies-literature

Engineering an Enzyme for Direct Electrical Monitoring of Activity.

Project description:Proteins have been shown to be electrically conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as sensors, detectors, and sequencing devices. We have engineered contact points into a ?29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. ?29 connected by one biotinylated contact, and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open to closed conformational transition of the polymerase. Polymerase activity is manifested by a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

| S-EPMC7047563 | biostudies-literature

Engineering enzyme activity using an expanded amino acid alphabet.

Project description:Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

| S-EPMC9863031 | biostudies-literature

Understanding single enzyme activity via the nano-impact technique.

Project description:To evaluate the possible detection of single enzyme activity via electrochemical methods, a combined finite difference and random walk simulation is used to model individual enzyme-electrode collisions where such events are monitored amperometrically via the measurement of products formed by the enzyme in solution. It is found that the observed signal is highly sensitive to both the enzyme turnover number, the size of the electrode and the bandwidth of the electronics. Taking single catalase impacts as an example, simulation results are compared with experimental data. Our work shows the requirement for the detection of electrochemically active product formed by individual enzymes and gives guidance for the design of experiments.

| S-EPMC5632796 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data