The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (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. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Elucidation of the biosynthesis of the methane catalyst coenzyme F430.

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA-CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

Exploring the onset of B12 -based mutualisms using a recently evolved Chlamydomonas auxotroph and B12 -producing bacteria.

Cobalamin (vitamin B12 ) 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 B12 . Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12 -dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12 -independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12 -dependent algae.

Plasmodium falciparum 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 Plasmodium falciparum, 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. In this study it is demonstrated that neither of these proteins possess UroS activity and the real UroS remains to be identified. This was demonstrated by the failure of codon-optimized genes to complement a defined Escherichia coli hemD- mutant (SASZ31) deficient in UroS activity. Furthermore, HPLC analysis of the oxidized reaction product from recombinant, purified P. falciparum HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, showing that P. falciparum HmbS does not have UroS activity and can only catalyze the formation of hydroxymethylbilane from porphobilinogen.

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. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

Replacement of the Cobalt Center of Vitamin B12 by Nickel: Nibalamin and Nibyric Acid Prepared from Metal-Free B12 †Ligands Hydrogenobalamin and Hydrogenobyric Acid.

The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transition-metal analogue of vitamin B12 . Described here is Nibl, synthesized by incorporation of a NiII ion into the metal-free B12  ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiII  corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal-free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X-ray analysis of Niby shows the structural adaptation of the corrin ligand to NiII ions and the coordination behavior of NiII . The diamagnetic Niby and Nibl, and corresponding isoelectronic CoI corrins, were deduced to be isostructural. Nibl is a structural mimic of four-coordinate base-off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase.

Zinc Substitution of Cobalt in Vitamin†B12 : Zincobyric acid and Zincobalamin as Luminescent Structural B12 -Mimics.

Replacing the central cobalt ion of vitamin B12 by other metals has been a long-held aspiration within the B12 -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 B12 , respectively. The solution structures of Znby and Znbl were studied by NMR-spectroscopy. Single crystals of Znby were produced, providing the first X-ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoII -corrins, making such Zn-corrins potentially useful for investigations of B12 -dependent processes. The singlet excited state of Znby had a short life-time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (ET =190 kJ mol-1 ) and was found to be an excellent photo-sensitizer for 1 O2 (ΦΔ =0.70).

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12 Cofactors for Catalysis.

The B12 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 B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single-crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B12 -cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12 .

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.

Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12.

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 Rh(III) than for Co(III) , challenging the current views concerning the evolution of corrins.

Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in α-proteobacteria.

It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.

The structure, function and properties of sirohaem decarboxylase--an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri AhbA/B have been produced and their physical properties compared. The D. vulgaris and M. barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D. desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed.

Characterization of the enzyme CbiH60 involved in anaerobic ring contraction of the cobalamin (vitamin B12) biosynthetic pathway.

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B(12)) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH(60), that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH(60) was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH(60) demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.

The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization.

The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe(2+) or Co(2+)) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX(S) from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.

Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12.

The acquisition of CoII by the corrin component of vitamin B12 follows one of two distinct pathways, referred to as early or late CoII insertion. The late insertion pathway exploits a CoII 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. In the metallochaperone-independent route, sirohydrochlorin (SHC) associates with the CbiK chelatase to form CoII-SHC. CoII-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for CoII transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form CoII-HBAD. Here, CoII-buffered enzymatic assays indicate that CoII transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for CoII binding. Notably, there is a favorable gradient for CoII transfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoII from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, CoII transfer from the chaperone to the chelatase complex is calculated to become favorable. These data reveal that the CobW metallochaperone can overcome an unfavorable thermodynamic gradient for CoII transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.

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

Vitamin B12, 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. In this chapter, we have outlined methods that we have found extremely helpful in the characterization of the biochemical pathway, including a plate microbiological assay, a corrinoid affinity extraction method, LCMS characterization and a multigene cloning strategy.

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 Bacteroides thetaiotaomicron contain multiple high-affinity vitamin B12 binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B12 belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability. We show that B. thetaiotaomicron derived BEVs bind a variety of cobamides and not only deliver them back to the parental bacterium but also sequester the micronutrient from competing bacteria. Additionally, Caco-2 cells, representing a model intestinal epithelial barrier, acquire cobamide-bound vesicles and traffic them to lysosomes, thereby mimicking the physiological cobalamin-specific intrinsic factor-mediated uptake process. Our findings identify a novel cobamide binding activity associated with BEVs with far-reaching implications for microbiota and host health.

Algae acquire vitamin B12 through a symbiotic relationship with bacteria.

Vitamin B12 (cobalamin) was identified nearly 80 years ago as the anti-pernicious anaemia factor in liver, and its importance in human health and disease has resulted in much work on its uptake, cellular transport and utilization. Plants do not contain cobalamin because they have no cobalamin-dependent enzymes. Deficiencies are therefore common in strict vegetarians, and in the elderly, who are susceptible to an autoimmune disorder that prevents its efficient uptake. In contrast, many algae are rich in vitamin B12, with some species, such as Porphyra yezoensis (Nori), containing as much cobalamin as liver. Despite this, the role of the cofactor in algal metabolism remains unknown, as does the source of the vitamin for these organisms. A survey of 326 algal species revealed that 171 species require exogenous vitamin B12 for growth, implying that more than half of the algal kingdom are cobalamin auxotrophs. Here we show that the role of vitamin B12 in algal metabolism is primarily as a cofactor for vitamin B12-dependent methionine synthase, and that cobalamin auxotrophy has arisen numerous times throughout evolution, probably owing to the loss of the vitamin B12-independent form of the enzyme. The source of cobalamin seems to be bacteria, indicating an important and unsuspected symbiosis.