Substrate channelling as an approach to cascade reactions

Wheeldon, Ian; Minteer, Shelley D.; Banta, Scott; Barton, Scott Calabrese; Atanassov, Plamen; Sigman, Matthew S.

doi:10.1038/nchem.2459

Cited by 541 publications

(551 citation statements)

References 113 publications

Supporting

Mentioning

532

Contrasting

Unclassified

Order By: Relevance

“…If respiratory-chain supercomplex assemblies contain the quinone/quinol substrate and channel it between their component enzymes, then a competing quinol oxidase, outside the supercomplex structure, should not be able to turn over (Bulutoglu et al., 2016, Wheeldon et al., 2016) (Figure 1). To test the effects of adding a competing quinol oxidase to mammalian respiratory membranes, we added 0.1 mg AOX per mg SMPs (hereafter written as 0.1 mg AOX mg −1 ) directly to a suspension of SMPs while monitoring the rate of NADH oxidation (NADH:O 2 oxidoreduction).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

2018

View full text Add to dashboard Cite

SummaryMitochondrial respiratory supercomplexes, comprising complexes I, III, and IV, are the minimal functional units of the electron transport chain. Assembling the individual complexes into supercomplexes may stabilize them, provide greater spatiotemporal control of respiration, or, controversially, confer kinetic advantages through the sequestration of local quinone and cytochrome c pools (substrate channeling). Here, we have incorporated an alternative quinol oxidase (AOX) into mammalian heart mitochondrial membranes to introduce a competing pathway for quinol oxidation and test for channeling. AOX substantially increases the rate of NADH oxidation by O2 without affecting the membrane integrity, the supercomplexes, or NADH-linked oxidative phosphorylation. Therefore, the quinol generated in supercomplexes by complex I is reoxidized more rapidly outside the supercomplex by AOX than inside the supercomplex by complex III. Our results demonstrate that quinone and quinol diffuse freely in and out of supercomplexes: substrate channeling does not occur and is not required to support respiration.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Introduction of an external enzyme to compete for a potentially channeled substrate is a diagnostic test for channeling (Bulutoglu et al., 2016, Wheeldon et al., 2016). If the substrate is truly channeled, flux through the competing pathway is negligible.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

2018

View full text Add to dashboard Cite

show abstract

“…It is evident from natural examples that compartmentalized reaction systems offer advantages when intermediate species are prone to escape into competing reaction channels [3][4][5][6][7][8] . The GDH-ST/SC-LbADH hydrogels can be used to investigate this phenomenon because the NADPH consuming LbADH reduction is spatially and kinetically coupled to the NADPH regeneration by GDH.…”

Section: Resultsmentioning

confidence: 99%

“…Towards the goal of the efficient use of renewable biomass as an alternative to petrochemical synthesis for sustainable production processes and energy supply in the future, multistep enzymatic cascade reactions are currently attracting much attention [3][4][5][6][7][8] . Such cascades are abundant in living systems, for instance, to maintain the regulation of metabolic activity or signal transduction [9][10][11] .…”

mentioning

confidence: 99%

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Peschke

Gallus

Bitterwolf

et al. 2017

Preprint

View full text Add to dashboard Cite

______________________________________________________________We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported. ______________________________________________________________

show abstract

“…This demonstrates that flux through the entire system was more efficient for the tethered enzymes. In biological systems, several mechanis ms of substrate channeling have been described [10] , with demonstrations that intermediates can move within the hydration shells around adjacent proteins, and that other intervening proteins can provide a hydration bridge [3a-c, 11] . Such models are highly compatible with our data, in which multiple enzymes were tethered to single NPs.…”

mentioning

confidence: 99%

Biomimicry Promotes the Efficiency of a 10‐Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate

et al. 2016

View full text Add to dashboard Cite

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. KeywordsBiomimicry; Enzymes; Glycolysis; Nanoparticles; Tethered Enzymes Multiple strategies have been used to interface biological components with inorganic surfaces. In engineering studies and industrial applications, carboxyl-amine binding chemistry is the most common attachment strategy [1] . However, this chemistry is not sitespecific and does not inherently impart proper molecular orientation-two factors that can * ajt32@cornell.edu.Supporting information for this article is given via a link at the end of the document. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript lead to significant reduction in function of the tethered enzyme [2] . Use of self-assembly templates can immobilize and orient enzymes; for instance sequential reactions of glucose oxidase and horseradish peroxidase have been demonstrated using a DNA scaffold to control enzyme spacing [3] . Recently, the sequential reaction of three enzymes conjugated to quantum dots was demonstrated [4] . Despite the different approaches available, the current state of the art remains limited to two-or three-step, coupled tethered reactions [5] . This is in stark contrast to multi-step reactions in solution, in which complex reactions have been demonstrated ranging up to a 13-step production of ethanol from glucose [6] .Inspired by the organization of glycolytic enzymes on the fibrous sheath of mammalian sperm [7] , we previously showed that replacement of germ cell-specific targeting domains with a bioaffinity tag provided oriented immobilization of glycolytic enzymes. This translated into significant advantages in specific activity of both individual enzymes and for sequential reactions when compared against the same enzymes tethered via random adsorption without a histidine tag or carboxyl-amine chemistry [8] These prior studies suggested that biomimetic oriented immobilization increased the activity of individual tethered enzymes for sev...

show abstract

Substrate channelling as an approach to cascade reactions

Cited by 541 publications

References 113 publications

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Biomimicry Promotes the Efficiency of a 10‐Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate

Contact Info

Product

Resources

About