We have constructed a synthetic mimic of the carboxysome, a cyanobacterial carbon-fixing organelle. Using an electrostatic tagging system, we coencapsulated the two key carboxysomal enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA), in an engineered protein cage based on lumazine synthase. A statistically significant kinetic effect of coencapsulated CA on RuBisCO activity was not observed under ambient or oxygen saturated conditions, suggesting that enzyme proximity alone may not be the key determinant in carboxysome function. The capsid shell protected the enzyme from proteolytic damage, a factor that could have provided early cyanobacteria with an evolutionary benefit. Our strategy to coencapsulate different proteins can easily be extended to other sequentially acting enzymes and lays down principles for developing artificial organelles to control biosynthetic pathways in vivo.

The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

Modification of aliphatic C−H bonds in a regio-and stereoselective manner can pose a formidable challenge in organic chemistry. In this context, the use of nonheme iron and α-ketoglutarate-dependent dioxygenases (αKGDs) represents an interesting tool for C−H activation as this enzyme family can catalyze a broad set of synthetically valuable reactions including hydroxylations, oxidations, and desaturations. The consensus reaction mechanism of αKGDs proceeds via the formation of a Fe(IV)-oxo complex capable of hydrogen atom transfer (HAT) from an sp 3 -hybridized substrate carbon center. The resulting substrate radical and Fe(III)−OH cofactor are considered to be the branch point toward the possible reaction outcomes which are determined by the enzyme's active site architecture. To date, the modulation of the reaction fate in Fe(II)/α-ketoglutarate-dependent dioxygenases via enzyme engineering has been mainly elusive. In this study, we therefore set out to engineer the L-proline cis-4-hydroxylase SmP4H from Sinorhizobium meliloti for selective oxidative modifications of the nonproteinogenic amino acid L-homophenylalanine (L-hPhe) to produce pharmacological relevant small molecule intermediates. Using structure-guided directed evolution, we improved the total turnover number, the k cat , as well as the k cat /K m of the hydroxylation reaction yielding the desired γ-hydroxylation product by approximately 10-fold, >100-fold, and >300-fold, respectively. Notably, the exchange of only one amino acid in the active site (W40Y) allowed us to reprogram the natural hydroxylase to predominantly act as a desaturase, presumably through tyrosine's capability to serve as a catalytic entity in the reaction mechanism. An investigation of the substrate scope revealed additional acceptance of the noncanonical amino acids Lhomotyrosine and (S)-α-amino-3,4-dichlorobenzenebutanoic acid by SmP4H variants.

Engineered variants of the capsid-forming enzyme lumazine synthase, AaLS, were used as nanoreactors for an enzyme-mediated polymerization. Oxidation of 3,3-diaminobenzidine (DAB) by the engineered ascorbate peroxidase APEX2 encapsulated in AaLS capsids resulted in templated formation of polyDAB-capsid nanoparticles of homogeneous size and shape.

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