Bacterial microcompartments (BMCs) are self-assembling organelles that consist of an enzymatic core that is encapsulated by a selectively permeable protein shell. The potential to form BMCs is widespread and found across the kingdom Bacteria. BMCs have crucial roles in carbon dioxide fixation in autotrophs and the catabolism of organic substrates in heterotrophs. They contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. Although BMCs were first visualized more than 60 years ago, it is mainly in the past decade that progress has been made in understanding their metabolic diversity and the structural basis of their assembly and function. This progress has not only heightened our understanding of their role in microbial metabolism but is also beginning to enable their use in a variety of applications in synthetic biology. In this Review, we focus on recent insights into the structure, assembly, diversity and function of BMCs.

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO₂-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B₁₂-dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer. Recently, BMCs associated with glycyl radical enzymes (GREs) were discovered; these are widespread and comprise at least three functionally distinct types. Previously, we predicted one type of these GRE-associated microcompartments (GRMs) represents a B₁₂-independent propanediol-utilizing BMC. Here we functionally and structurally characterize enzymes of the GRM of Rhodopseudomonas palustris BisB18 and demonstrate their concerted function in vitro. The GRM signature enzyme, the GRE, is a dedicated 1,2-propanediol dehydratase with a new type of intramolecular encapsulation peptide. It forms a complex with its activating enzyme and, in conjunction with an aldehyde dehydrogenase, converts 1,2-propanediol to propionyl-CoA. Notably, homologous GRMs are also encoded in pathogenic Escherichia coli strains. Our high-resolution crystal structures of the aldehyde dehydrogenase lead to a revised reaction mechanism. The successful in vitro reconstitution of a part of the GRM metabolism provides insights into the metabolic function and steps in the assembly of this BMC.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO₂) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO₂ molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO₂ within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO₂-fixing enzyme. Together, these 4 residues anchor and position the CO₂ molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO₂ fixation. Altogether, our study reveals unprecedented molecular details of selective CO₂ binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO₂-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO₂ in biology and chemistry.