Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers

Plegaria, Jefferson S.; Yates, Matthew D.; Glaven, Sarah M.; Kerfeld, Cheryl A.

doi:10.1021/acsabm.9b01023

Cited by 11 publications

(6 citation statements)

References 57 publications

(115 reference statements)

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“…The new function of this enzyme in BMCs also established the BMC shell as a separating membrane for cofactor pools and conduit for electrons. This extends previous work on engineering metal centers into the pores of BMC shell proteins 53,54 and sets the stage for connecting redox functions between the inside and outside of BMC shells.…”

Section: Discussionsupporting

confidence: 72%

Electrochemical cofactor recycling of bacterial microcompartments

Sutter,

Utschig,

Niklas

et al. 2024

Preprint

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Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSEassists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

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Section: Discussionsupporting

confidence: 72%

Electrochemical cofactor recycling of bacterial microcompartments

Sutter,

Utschig,

Niklas

et al. 2024

Preprint

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“…The redox potential of the cluster could also be tuned by changing the position of the coordinating cysteine. 118 Appropriately positioned cysteines can also occlude pores by forming disulfide bonds under oxidizing conditions, a phenomenon that has been shown in a PduA-S40C mutant. 34 4.2.5.…”

Section: Pore Engineering: Methodologies Andmentioning

confidence: 91%

“…This engineered shell was able to reversibly cycle between reduced and oxidized states with the cluster remaining intact. The redox potential of the cluster could also be tuned by changing the position of the coordinating cysteine . Appropriately positioned cysteines can also occlude pores by forming disulfide bonds under oxidizing conditions, a phenomenon that has been shown in a PduA-S40C mutant …”

Section: Pore Engineering: Methodologies and Applicationsmentioning

confidence: 99%

How Pore Architecture Regulates the Function of Nanoscale Protein Compartments

et al. 2022

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Self-assembling proteins can form porous compartments that adopt well-defined architectures at the nanoscale. In nature, protein compartments act as semipermeable barriers to enable spatial separation and organization of complex biochemical processes. The compartment pores play a key role in their overall function by selectively controlling the influx and efflux of important biomolecular species. By engineering the pores, the functionality of compartments can be tuned to facilitate non-native applications, such as artificial nanoreactors for catalysis. In this review, we analyze how protein structure determines the porosity and impacts the function of both native and engineered compartments, highlighting the wealth of structural data recently obtained by cryo-EM and X-ray crystallography. Through this analysis, we offer perspectives on how current structural insights can inform future studies into the design of artificial protein compartments as nanoreactors with tunable porosity and function.

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“…[14,100] Protein engineering has also modified BMC shell proteins for specific functions. [101,102] For example, introducing natural and/or engineered metal-ion coordinating BMC shell proteins could facilitate electron transfer, [103][104][105] while BMC shell proteins with assembly protecting groups could enable protease responsive membrane formation, [20] and those with protein-protein adaptor domains would allow cargo scaffolding. [61,106,107] Understanding the dynamics of shell protein exchange in BMC shell protein systems will prove important for future studies.…”

Section: Discussionmentioning

confidence: 99%

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems

Abeysinghe,

Young,

Rowland

et al. 2023

Small

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Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self‐assembling bacterial microcompartment (BMC) shell proteins and liquid‐liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid‐liquid interfaces between either 1) the dextran‐rich droplets and PEG‐rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two‐phase system, or 2) the polypeptide‐rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically‐driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three‐phase system wherein coacervate droplets are contained within dextran‐rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three‐phase system by changing the polyelectrolyte charge ratio. The tens‐of‐micron scale BMC shell protein‐coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

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Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers

Cited by 11 publications

References 57 publications

Electrochemical cofactor recycling of bacterial microcompartments

Electrochemical cofactor recycling of bacterial microcompartments

How Pore Architecture Regulates the Function of Nanoscale Protein Compartments

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems

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