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Artificial metalloenzymes combine the inherent reactivity of transition metal catalysis with the sophisticated reaction control of natural enzymes. By providing new opportunities in bioorthogonal chemistry and biocatalysis, artificial metalloenzymes have the potential to overcome certain limitations in both drug discovery and green chemistry or related research fields. Ongoing advances in organometallic catalysis, directed evolution, and bioinformatics are enabling the design of increasingly powerful systems that outperform conventional catalysis in a growing number of cases. Therefore, this review article collects challenges and opportunities in designing artificial metalloenzymes described in recent review articles. This will provide an equitable insight for those new to and interested in the field.

In this study, we describe the creation of an artificial protein cage housing a dual metal-tagged guest protein that catalyzes a linear, two-step sequential cascade reaction. The guest protein consists of a fusion protein of HaloTag and monomeric rhizavidin. Inside the protein capsid, we establish a ruthenium-catalyzed alloc-deprotection followed by a gold-catalyzed ring-closing hydroamination reaction that leads to indoles and phenanthridines with an overall yield of up to 67% in aqueous solutions. Furthermore, we show that the encapsulation stabilizes the metal catalysts against deactivation by air, proteins and cell lysate.

Spatial control over chemical reactions is a ubiquitous feature shared by all living organisms. In the present article, we describe the design of a proteinaceous, synthetic capsid based on the well-described bacterial nanocompartment encapsulin. Our engineered virus-like particle carries a dual functional artificial metalloenzyme (ArM) as non-native guest protein. This ArM, created from a fusion protein of HaloTag and monomeric rhizavidin, serves as catalyst for a fully bioorthogonal, linear, two-step reaction cascade. A ruthenium-catalyzed alloc deprotection is followed by a gold-catalyzed, ring-closing hydroamination reaction leading to indoles and phenanthridines with up to 67 % overall yield in aqueous solutions.

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

Due to the emergence of multidrug resistant bacteria, the development of new antibiotics is required. We introduce here asymmetrically modified positively charged bis(methylpyridinium) anthracenes as a novel tunable scaffold, in which the two positive charges can be placed at a defined distance and angle. Our structure-activity relationship reveals that coupling the methylpyridiniums with alkynyl linkers to the central anthracene unit yields antibacterial compounds against a wide range of bacteria, including Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. Also, different mycobacteria, such as Mycobacterium smegmatis and Mycobacterium tuberculosis, are efficiently targeted by these compounds. The antibacterial activity depends on the number of alkynyl linkers and consequently also on the distance of the positive charges in the rigid anthracene scaffold. Additionally, the formation of an anthracene endoperoxide further increases the antibacterial activity, likely due to the release of toxic singlet oxygen that converts the endoperoxide back to the antibacterial anthracene scaffold with half-lives of several hours.

A tunable chemoenzymatic strategy provides access to the entire class of magic spot nucleotides and modified analogues. The approach combines chemoselective bisphosphorylations using phosphoramidites with regioselective ribonuclease T2 cyclo-phosphate hydrolysis, leading to flexible and simple gram-scale operations.

In this study, we report that Streptomyces asterosporus DSM 41452 is a producer of new molecules related to the nonribosomal cyclodepsipeptide WS9326A and the polyketide annimycin. S. asterosporus DSM 41452 is shown to produce six cyclodepsipeptides and peptides, WS9326A to G. Notably, the compounds WS9326F and WS9326G have not been described before. The genome of S. asterosporus DSM 41452 was sequenced, and a putative WS9326A gene cluster was identified. Gene-deletion experiments confirmed that this cluster was responsible for the biosynthesis of WS9326A to G. Additionally, a gene-deletion experiment demonstrated that sas16 encoding a cytochrome P450 monooxygenase was involved in the synthesis of the novel (E)-2,3-dehydrotyrosine residue found in WS9326A and its derivatives. An insertion mutation within the putative annimycin gene cluster led to the production of a new annimycin derivative, annimycin B, which exhibited modest inhibitory activity against Plasmodium falciparum.

The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors that have reached clinical development. These two new compounds were characterized by an array of cell-based assays and gene expression profiling experiments. A global chemical proteomics approach was used to generate the drug-protein interaction profile, which suggested suitable therapeutic indications. Both inhibitors, studied in the context of angiogenesis and in vivo in a relevant lymphoma model, showed high efficacy in the control of tumor size.

Summary Nutrient uptake is essential for survival of organisms, and carbohydrates serve as a crucial carbon and energy source for most microorganisms. Given the importance of mycobacteria as human pathogens a detailed knowledge of carbohydrate uptake transporters is highly desirable, but currently available information is severely limited and mainly based on in silico analyses. Moreover, there is only very little data available on the in vitro characterization of carbohydrate transporters from mycobacterial species. To overcome these significant limitations there is a strong demand for innovative approaches to experimentally match substrates to ATP-binding cassette (ABC) transporters in a straightforward manner. Our study focuses on the model organism Mycobacterium smegmatis and identifies a mycobacterial ABC transport system based on a rapid label-free mass spectrometry lipoproteomics assay with broad applicability. Further validation and X-ray structure analyses reveal a highly selective mycobacterial L-arabinose uptake system.

Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A–D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.

Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A–D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self‐assembling protein‐based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single‐molecule Förster resonance energy transfer and 3D‐MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single‐molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non‐native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space., A bioorthogonal catalysis strategy has been developed inside a confined space using encapsulins equipped with HaloTag and [CpRu(hydroxyquinoline)(allyl)]PF6. The strategy opens up a toolbox for designing compartmentalized “new‐to‐biology” reactions inside living cells.