Enzyme-mediated polymerization inside engineered protein cages

Frey, Raphael; Hayashi, Takahiro; Hilvert, Donald

doi:10.1039/c6cc05301g

Cited by 36 publications

(28 citation statements)

References 34 publications

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“…17 +36GFP could also be used as a fusion tag, which when appended to a number of different enzymes was able to direct their encapsulation. [18][19][20][21][22] Amongst these, an engineered ascorbate peroxidase (APEX2) was proven to perform constrained polymerization of compound 3,3- diaminobenzidine (DAB) inside the LS cage 20 while encapsulation of Tobacco Etch Virus (TEV) protease allowed construction of a substrate-sorting nanoreactor (Fig. 1b).…”

Section: Lumazine Synthasementioning

confidence: 99%

Enzyme encapsulation by protein cages

et al. 2020

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Section: Lumazine Synthasementioning

confidence: 99%

Enzyme encapsulation by protein cages

et al. 2020

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“…14,17,18 The high affinity of this cationic protein for the lumen of AaLS-13 had made it a practical packaging tag for targeting diverse enzymes to the cage interior. 19−21 Here we exploit this strategy to encapsulate a sequence-specific protease from Tobacco Etch virus (TEV) and show that the resulting proteolytic nanoreactor also sorts potential substrates according to their net charge, promoting uptake and hydrolysis of positively charged peptides and proteins while excluding negatively charged competitors (Figure 1a).…”

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confidence: 99%

Substrate Sorting by a Supercharged Nanoreactor

2018

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Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts.

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“…Because the enzyme is readily produced in recombinant form, exhibits extraordinary thermostability (T m ϳ120°C) (19), and tolerates both chemical and genetic modifi-cation, AaLS mutants have been widely utilized for biomimetic applications. These include biomineralization (28), drug delivery (29,30), bioimaging (31), nucleic acid storage (32), enzyme catalysis (33), templated synthesis of polymers (34), and mimicry of bacterial microcompartments like the carboxysome (35). Identification of a specific peptide localization sequence that enables encapsulation of guest molecules by native AaLS cages lays the groundwork for investigating the nature of RS⅐LS complexation, as well as for further expanding the range of biotechnological applications of this system.…”

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The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase

Azuma¹,

Zschoche²,

Hilvert

2017

Journal of Biological Chemistry

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Encapsulation of specific enzymes in self-assembling protein cages is a hallmark of bacterial compartments that function as counterparts to eukaryotic organelles. The cage-forming enzyme lumazine synthase (LS) from (BsLS), for example, encapsulates riboflavin synthase (BsRS), enabling channeling of lumazine from the site of its generation to the site of its conversion to vitamin B Elucidating the molecular mechanisms underlying the assembly of these supramolecular complexes could help inform new approaches for metabolic engineering, nanotechnology, and drug delivery. To that end, we investigated a thermostable LS from (AaLS) and found that it also forms cage complexes with the cognate riboflavin synthase (AaRS) when both proteins are co-produced in the cytosol of A 12-amino acid-long peptide at the C terminus of AaRS serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Sequence comparisons suggested that analogous peptide segments likely direct RS complexation by LS cages in other bacterial species. Covalent fusion of this peptide tag to heterologous guest molecules led to their internalization into AaLS assemblies both and, providing a firm foundation for creating tailored biomimetic nanocompartments for medical and biotechnological applications.

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Enzyme-mediated polymerization inside engineered protein cages

Cited by 36 publications

References 34 publications

Enzyme encapsulation by protein cages

Enzyme encapsulation by protein cages

Substrate Sorting by a Supercharged Nanoreactor

The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase

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