55 research outputs found
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
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 hemDâ 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
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 coenzymeâ
B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12âdependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5â˛âdeoxyâ5â˛âadenosylrhodibalamin (AdoRbl). AdoRbl was prepared by deâ
novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive inâ
vivo in microbial bioassays for methionine synthase and acted as an inâ
vitro inhibitor of an AdoCblâdependent 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
Replacement of the Cobalt Center of Vitamin B 12 by Nickel: Nibalamin and Nibyric Acid Prepared from MetalâFree B 12 Ligands Hydrogenobalamin and Hydrogenobyric Acid
The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transitionâmetal analogue of vitaminâ
B12. Described here is Nibl, synthesized by incorporation of a NiII ion into the metalâfree B12â
ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiIIâ
corrin nibyric acid (Niby) was similarly synthesized from Hby, the metalâfree 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. Xâray 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 fourâcoordinate baseâoff Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase
Human Intrinsic Factor Expression for Bioavailable Vitamin B12 Enrichment in Microalgae
Dietary supplements and functional foods are becoming increasingly popular complements to regular diets. A recurring ingredient is the essential cofactor vitamin B12 (B12). Microalgae are making their way into the dietary supplement and functional food market but do not produce B12, and their B12 content is very variable. In this study, the suitability of using the human B12-binding protein intrinsic factor (IF) to enrich bioavailable B12 using microalgae was tested. The IF protein was successfully expressed from the nuclear genome of the model microalga Chlamydomonas reinhardtii and the addition of an N-terminal ARS2 signal peptide resulted in efficient IF secretion to the medium. Co-abundance of B12 and the secreted IF suggests the algal produced IF protein is functional and B12-binding. Utilizing IF expression could be an efficient tool to generate B12-enriched microalgae in a controlled manner that is suitable for vegetarians and, potentially, more bioavailable for humans
Synthesis, spectral characterization and crystal structure of Chlororhodibalamin: A synthesis platform for rhodium analogues of vitamin B12 and for Rh-based antivitamins B12
Chlororhodibalamin (ClRhbl), a rhodium analogue of vitamin B12Â (cyanocobalamin), was prepared in 84% yield by metalation of the metal-free B12Â ligand hydrogenobalamin using the RhI-complex [Rh(CO)2Cl]2. ClRhbl was identified and characterized by UV/Vis, circular dichroism, high-resolution mass and heteronuclear NMR spectra. The RhIII-corrin ClRhbl features the 'base-on' architecture of vitamin B12. X-ray analysis of single crystals of ClRhbl have revealed its detailed 3D-geometry and close structural similarity to the CoIII-analogue chlorocobalamin (ClCbl). ClRhbl is a versatile starting material for the preparation of other rhodibalamins, among them the organometallic derivatives adenosylrhodibalamin and methylrhodibalamin, the Rh analogues of the important coenzyme and cofactor forms of B12, adenosylcobalamin and methylcobalamin
Solution, Crystal and in Silico Structures of the Organometallic Vitamin B 12 âDerivative Acetylcobalamin and of its Novel RhodiumâAnalogue Acetylrhodibalamin
The natural vitamin B12âderivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ringâcontracted corrin ligand. Here, we describe the synthesis of the Rh(III)âcorrin acetylrhodibalamin (AcRhbl) from biotechnologically produced metalâfree hydrogenobyric acid and analyze the effect of the replacement of the cobaltâcenter of the organometallic vitamin B12âderivative acetylcobalamin (AcCbl) with its groupâIX homologue rhodium, to give AcRhbl. The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotideâappended corrinâligand in these two metal corrins features extensive structural similarity. Thus, the rhodiumâcorrin AcRhbl joins the small group of B12âmimics classified as âantivitamins B12â, isostructural metal analogues of the natural cobaltâcorrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes
The rhodium analogue of Coenzyme B 12 as an antiâphotoregulatory ligand inhibiting bacterial CarH photoreceptors
Coenzyme B12 (AdoCbl; 5â˛âdeoxyâ5â˛âadenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The lightâresponsive cobaltâcorrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNAâbinding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCblâbound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the nonânatural 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 âbaseâoff/Hisâonâ to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented antiâphotoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCblâbased 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 nonâradical synâ1,2âelimination mechanism, an aspect not previously considered for this photoreceptor
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 longâheld aspiration within the B12âfield. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Znâanalogues of the natural cobaltâcorrins cobyric acid and vitaminâ
B12, respectively. The solution structures of Znby and Znbl were studied by NMRâspectroscopy. Single crystals of Znby were produced, providing the first Xâray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoIIâcorrins, making such Znâcorrins potentially useful for investigations of B12âdependent processes. The singlet excited state of Znby had a short lifeâtime, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (ET=190â
kJâmolâ1) and was found to be an excellent photoâsensitizer for 1O2 (ÎŚÎ=0.70)
The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12âCofactors 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 de-symmetrized âentaticâ 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 analogs of B12
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