Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.It has been recognized for more than 30 years that all cyanobacteria (1) and some other chemoautotrophic bacteria (2) contain carboxysomes. These polyhedral cellular inclusions consist of a proteinaceous shell enclosing an active enzyme, ribulose bisphosphate carboxylase/oxygenase (RuBisCO). 4Their function is to enhance the fixation of carbon dioxide (3), a reaction of planetary significance in that marine cyanobacteria are responsible for the majority of global carbon fixation (4, 5). More recently, sequence similarity was noticed between carboxysome shell genes and metabolic operon genes associated with propanediol utilization (pdu) and ethanolamine utilization (eut) in a variety of heterotrophic bacteria found in the mammalian gut (3) and the environment. In growth conditions that induce these metabolic operons, polyhedral organelles resembling carboxysomes were observed on electron microscopy of Salmonella enterica serovar Typhimurium (6), Klebsiella oxytoca, Citrobacter freundii, and Escherichia coli (7). Bioinformatics analysis also locates genes resembling carboxysome shell genes in metabolic operons in Clostridium perfringens (8), Clostridium tetani (9), Listeria monocytogenes and Listeria innocua (10), Enterococcus faecalis (11), Lactobacillus collinoides (12), Citrobacter rodentium, 5 and Yersinia enterocolitica (13) among other organisms. The non-carboxysome polyhedral structures have been referred to as enterosomes (3) or metabolosomes (14), emphasizing their role in cellular metabolism.There is some considerable interest in how these proteinaceous organelles form and the arrangement of protein subunits that give rise to these remarkable macromolecular assemblies. In carboxysomes, there are thought to be a number of shell prot...

It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.

B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12‐dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5′‐deoxy‐5′‐adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl‐dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for RhIII than for CoIII, challenging the current views concerning the evolution of corrins.

The aerobic biosynthetic pathway for vitamin B12 (cobalamin) biosynthesis is reviewed. Particular attention is focused on the ring contraction process, whereby an integral carbon atom of the tetrapyrrole-derived macrocycle is removed. Previous work had established that this chemically demanding step is facilitated by the action of a mono-oxygenase called CobG, which generates a hydroxy lactone intermediate. This mono-oxygenase contains both a non-haem iron and an Fe-S centre, but little information is known about its mechanism. Recent work has established that in bacteria such as Rhodobacter capsulatus, CobG is substituted by an isofunctional protein called CobZ. This protein has been shown to contain flavin, haem and Fe-S centres. A mechanism is proposed to explain the function of CobZ. Another interesting aspect of the aerobic cobalamin biosynthetic pathway is cobalt insertion, which displays some similarity to the process of magnesium chelation in chlorophyll synthesis. The genetic requirements of cobalt chelation and the subsequent reduction of the metal ion are discussed.

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