The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III.

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in , employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called , encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from () .

The Rhodium Analogue of Coenzyme B as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression.

Exploiting Differences in Heme Biosynthesis between Bacterial Species to Screen for Novel Antimicrobials.

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an YFP strain were replaced with those for the CPD pathway from (SA) through a sliding modular gene replacement recombineering strategy to generate the strain -CPD-YFP.

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans.

Modification of bacterial microcompartments with target biomolecules post-translational SpyTagging.

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein.

Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B.

The acquisition of Co by the corrin component of vitamin B follows one of two distinct pathways, referred to as early or late Co insertion. The late insertion pathway exploits a Co metallochaperone (CobW) from the COG0523 family of G3E GTPases, while the early insertion pathway does not. This provides an opportunity to contrast the thermodynamics of metalation in a metallochaperone-requiring and a metallochaperone-independent pathway.

Nutrient smuggling: Commensal gut bacteria-derived extracellular vesicles scavenge vitamin B12 and related cobamides for microbe and host acquisition.

The processes by which bacteria proactively scavenge essential nutrients in crowded environments such as the gastrointestinal tract are not fully understood. In this context, we observed that bacterial extracellular vesicles (BEVs) produced by the human commensal gut microbe contain multiple high-affinity vitamin B binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability.

Getting the Payload in Place - Unravelling the Complexities of Making a Bacterial Microcompartment.

Bacterial microcompartments (BMCs) are complex macromolecular assemblies composed of any outer protein shell that encases a specific metabolic pathway cargo. Recent research is now starting to unravel some of the processes that are involved in directing the enzyme cargo to the inside of the BMC. In particular, an article in this issue of J Bacteriol by N.

Exploring the onset of B -based mutualisms using a recently evolved Chlamydomonas auxotroph and B -producing bacteria.

Cobalamin (vitamin B ) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B . Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence.

Biosynthesis of cobamides: Methods for the detection, analysis and production of cobamides and biosynthetic intermediates.

Vitamin B, cobalamin, belongs to the broader cobamide family whose members are characterized by the presence of a cobalt-containing corrinoid ring. The ability to detect, isolate and characterize cobamides and their biosynthetic intermediates is an important prerequisite when attempting to study the synthesis of this remarkable group of compounds that play diverse roles across the three kingdoms of life. The synthesis of cobamides is restricted to only certain prokaryotes and their structural complexity entails an equally complex synthesis orchestrated through a multi-step biochemical pathway.

hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway.

Uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in , but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). Additionally, an unknown protein encoded by PF3D7_1247600 has also been predicted to possess UroS activity.

Calculating metalation in cells reveals CobW acquires Co for vitamin B biosynthesis while related proteins prefer Zn.

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells.

The requirement for cobalt in vitamin B: A paradigm for protein metalation.

Vitamin B, cobalamin, is a cobalt-containing ring-contracted modified tetrapyrrole that represents one of the most complex small molecules made by nature. In prokaryotes it is utilised as a cofactor, coenzyme, light sensor and gene regulator yet has a restricted role in assisting only two enzymes within specific eukaryotes including mammals. This deployment disparity is reflected in another unique attribute of vitamin B in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities.

Replacement of the Cobalt Center of Vitamin B by Nickel: Nibalamin and Nibyric Acid Prepared from Metal-Free B Ligands Hydrogenobalamin and Hydrogenobyric Acid.

The (formal) replacement of Co in cobalamin (Cbl) by Ni generates nibalamin (Nibl), a new transition-metal analogue of vitamin B . Described here is Nibl, synthesized by incorporation of a Ni ion into the metal-free B  ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related Ni  corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal-free cobyric acid ligand.

Biosynthesis of the modified tetrapyrroles-the pigments of life.

Modified tetrapyrroles are large macrocyclic compounds, consisting of diverse conjugation and metal chelation systems and imparting an array of colors to the biological structures that contain them. Tetrapyrroles represent some of the most complex small molecules synthesized by cells and are involved in many essential processes that are fundamental to life on Earth, including photosynthesis, respiration, and catalysis. These molecules are all derived from a common template through a series of enzyme-mediated transformations that alter the oxidation state of the macrocycle and also modify its size, its side-chain composition, and the nature of the centrally chelated metal ion.

Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

Metabolosomes, catabolic bacterial microcompartments (BMCs), are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multicomponent protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation before the formation of the outer shell.

Zinc Substitution of Cobalt in Vitamin B : Zincobyric acid and Zincobalamin as Luminescent Structural B -Mimics.

Replacing the central cobalt ion of vitamin B by other metals has been a long-held aspiration within the B -field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn-analogues of the natural cobalt-corrins cobyric acid and vitamin B , respectively. The solution structures of Znby and Znbl were studied by NMR-spectroscopy.

Bacterial Microcompartment-Mediated Ethanolamine Metabolism in Escherichia coli Urinary Tract Infection.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal , both pathogenic enterohemorrhagic and commensal strains. During a UTI, urease-negative bacteria thrive, despite the comparative nutrient limitation in urine.

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B Cofactors for Catalysis.

The B cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign.

Bacterial sensors define intracellular free energies for correct enzyme metalation.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define.

Biotechnological Advances in Bacterial Microcompartment Technology.

Bacterial microcompartments (BMCs) represent proteinaceous macromolecular nanobioreactors that are found in a broad range of bacteria, and which are associated with either anabolic or catabolic processes. They consist of a semipermeable outer shell that packages a central metabolic enzyme or pathway, providing both enhanced flux and protection against toxic intermediates. Recombinant production of BMCs has led to their repurposing with the incorporation of altogether new pathways.

De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments.

Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation.

Staphylococcus aureus haem biosynthesis and acquisition pathways are linked through haem monooxygenase IsdG.

Haem is an essential cofactor in central metabolic pathways in the vast majority of living systems. Prokaryotes acquire haem via haem biosynthesis pathways, and some also utilize haem uptake systems, yet it remains unclear how they balance haem requirements with the paradox that free haem is toxic. Here, using the model pathogen Staphylococcus aureus, we report that IsdG, one of two haem oxygenase enzymes in the haem uptake system, inhibits the formation of haem via the internal haem biosynthesis route.