Does metabolite channeling accelerate enzyme-catalyzed cascade reactions?

Poshyvailo, Liubov; Lieres, Eric von; Kondrat, Svyatoslav

doi:10.1371/journal.pone.0172673

Cited by 44 publications

(38 citation statements)

References 61 publications

(61 reference statements)

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“…The advantage of such scaffolding systems is that they can be used to direct and align biosynthetic pathway enzymes to orchestrate greater production of commodity and speciality chemicals, especially in pathways that proceed through unstable or toxic intermediates 6,7 . This is because the close proximity of enzymes on a scaffold allow for greater channeling of intermediates, through improved flux, stabilization of intermediates and protection from other reactions 8,9 .…”

Section: Engineered Bacterial Cytoscaffoldsmentioning

confidence: 99%

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

et al. 2017

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We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

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Section: Engineered Bacterial Cytoscaffoldsmentioning

confidence: 99%

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

et al. 2017

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“…Inspired by the cascade processes in nature, scientists have recently developed effective strategies in order to artificially co-localize diverse enzymes on nanocarriers for the development of nanobiocatalytic systems [20,21]. Multi-enzyme immobilization places several free enzymes in proximity, thus enabling the substrate channeling effect that can remarkably reduce diffusion losses and accelerate the overall cascade reaction rate [22][23][24]. So far, the successful simultaneous covalent co-immobilization of various classes of enzymes, mainly hydrolases and oxidoreductases, on magnetic nanoparticles for the catalysis of cascade reactions of biotechnological interest has been reported.…”

Section: Introductionmentioning

confidence: 99%

Development of a Four-Enzyme Magnetic Nanobiocatalyst for Multi-Step Cascade Reactions

et al. 2019

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We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 °C and remained active even after 24 d of incubation at 5 °C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose.

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“…Substrate channeling is most frequently described as a physiological mechanism that leads to efficient utilization of reactive metabolites 23,29,39 . The concentration of glycolytic enzymes in cells is much higher than the concentration of their substrates 23 , what indicates that in glycolysis the regulation of enzyme activity can be more important than efficient substrate utilization 11,47 . Our results indicate that substrate channeling can regulate metabolism by at least two different mechanisms: i ) allosteric regulation of differences in enzyme-substrate off -rates between different substrates or isozymes (supp.…”

Section: Discussionmentioning

confidence: 99%

Substrate Channelingviaa Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

Svedružić

Odorčić

Chang

et al. 2020

Preprint

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Background: D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADHchanneling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells. Results: Enzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)channeling between the adjacent subunits in each tetramer. Conclusions: In the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between off-rates for the transient (GAPDH-NADH)-LDH complex and off-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths. Highlights-Substrate channeling molecular mechanism can regulate energy production and aerobic and anaerobic metabolism in cells -LDH and GAPDH can form a channeling complex only at sub-saturating NADH concentration -Channeled and diffusive paths always compete and take place in parallel -NADH channeling does not require a high-affinity complex -NADH channeling within GAPDH-LDH complex is an extension of NAD(H) channeling within each tetramer -Allosteric modulation of NADH binding affinity in GAPDH tetramer can regulate NAD(H) channeling Introduction D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenases (LDH) are two NAD(H) dependent dehydrogenases that participate in glycolytic and gluconeogenic pathways 1,2 . Glycolysis and gluconeogenesis are the two major metabolic pathways that provide cells with metabolic precursors and a rapid source of energy. Parallel to glycolysis GAPDH participates in microtubule bundling, DNA replication and repair, apoptosis, the export of nuclear RNA, membrane fusion, and phosphotransferase activity 1,2 . GAPDH is implicated in Huntington's disease 3 , prostate cancer, and viral pathogenesis 1,2 . GAPDH could be a target of nitric oxide 4 and a target of ...

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Does metabolite channeling accelerate enzyme-catalyzed cascade reactions?

Cited by 44 publications

References 61 publications

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Development of a Four-Enzyme Magnetic Nanobiocatalyst for Multi-Step Cascade Reactions

Substrate Channelingviaa Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

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