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1.
Angew Chem Int Ed Engl ; 63(18): e202401626, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38416546

RESUMO

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.


Assuntos
Fosfotreonina/análogos & derivados , Ródio , Ligantes , Cobamidas/química , Bactérias/metabolismo , DNA
2.
JACS Au ; 3(5): 1472-1483, 2023 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-37234125

RESUMO

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.

3.
Methods Enzymol ; 668: 3-23, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589198

RESUMO

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.


Assuntos
Cobamidas , Vitamina B 12 , Cobamidas/química , Cobamidas/metabolismo , Vitamina B 12/química
4.
Environ Microbiol ; 24(7): 3134-3147, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35593514

RESUMO

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.


Assuntos
Chlamydomonas , Simbiose , Bactérias/genética , Chlamydomonas/genética , Chlamydomonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mesorhizobium , Vitamina B 12/genética , Vitamina B 12/metabolismo
5.
J Extracell Biol ; 1(10): e61, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38939214

RESUMO

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.

6.
Microbiology (Reading) ; 167(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34661520

RESUMO

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.


Assuntos
Heme/biossíntese , Hidroximetilbilano Sintase/metabolismo , Plasmodium falciparum/enzimologia , Vias Biossintéticas , Escherichia coli/genética , Teste de Complementação Genética , Hidroximetilbilano Sintase/genética , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Uroporfirinogênio III Sintetase/genética , Uroporfirinogênio III Sintetase/metabolismo , Uroporfirinogênios/metabolismo
7.
Nat Commun ; 12(1): 1195, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33608553

RESUMO

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. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Vitamina B 12/biossíntese , Zinco/metabolismo , Escherichia coli , GTP Fosfo-Hidrolases , Metais/metabolismo , Salmonella
8.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118896, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33096143

RESUMO

Vitamin B12, 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 B12 in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities. The core component of cobalamin is the corrin macrocycle that acts as the main ligand for the cobalt. Within this review we investigate why cobalt is paired specifically with the corrin ring, how cobalt is inserted during the biosynthetic process, how cobalt is made available within the cell and explore the cellular control of cobalt and cobalamin levels. The partitioning of cobalt for cobalamin biosynthesis exemplifies how cells assist metalation.


Assuntos
Cobalto/metabolismo , Simbiose/genética , Tetrapirróis/química , Vitamina B 12/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Cobalto/química , Coenzimas/genética , Coenzimas/metabolismo , Corrinoides/genética , Humanos , Ligantes , Tetrapirróis/metabolismo , Vitamina B 12/química , Vitamina B 12/genética
9.
Angew Chem Int Ed Engl ; 59(45): 20129-20136, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32686888

RESUMO

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.


Assuntos
Cobalto/química , Níquel/química , Vitamina B 12/química , Cristalografia por Raios X/métodos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Espectrofotometria Ultravioleta/métodos , Vitamina B 12/análogos & derivados
10.
Angew Chem Int Ed Engl ; 58(41): 14568-14572, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31420932

RESUMO

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).


Assuntos
Cobalto/química , Vitamina B 12/análogos & derivados , Vitamina B 12/química , Zinco/química , Luminescência , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Termodinâmica
11.
Angew Chem Int Ed Engl ; 58(31): 10756-10760, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31115943

RESUMO

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 .


Assuntos
Corrinoides/biossíntese , Uroporfirinas/metabolismo , Vitamina B 12/metabolismo , Biocatálise , Cobalto/química , Cobalto/metabolismo , Corrinoides/química , Ligantes , Estrutura Molecular , Uroporfirinas/química , Vitamina B 12/química
12.
Nat Chem Biol ; 15(3): 241-249, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30692683

RESUMO

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.


Assuntos
Transferência de Energia/fisiologia , Metaloproteínas/metabolismo , Metais/metabolismo , Marcadores de Afinidade/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Metaloproteínas/fisiologia , Salmonella/metabolismo
13.
Cell Chem Biol ; 25(8): 941-951.e6, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-29779954

RESUMO

Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms, and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogs with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogs were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms, and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogs, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B12.


Assuntos
Bactérias/metabolismo , Caenorhabditis elegans/metabolismo , Corantes Fluorescentes/metabolismo , Lepidium sativum/metabolismo , Vitamina B 12/metabolismo , Animais , Infecções Bacterianas/microbiologia , Transporte Biológico , Corrinoides/análise , Corrinoides/metabolismo , Corantes Fluorescentes/análise , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/análise
14.
Biology (Basel) ; 7(1)2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29463047

RESUMO

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 Chlamydomonasreinhardtii 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.

16.
Nature ; 543(7643): 78-82, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28225763

RESUMO

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.


Assuntos
Biocatálise , Vias Biossintéticas , Coenzimas/biossíntese , Metaloporfirinas/metabolismo , Metano/biossíntese , Methanosarcina barkeri/enzimologia , Tetrapirróis/biossíntese , Amidoidrolases/genética , Amidoidrolases/metabolismo , Vias Biossintéticas/genética , Coenzimas/química , Liases/genética , Liases/metabolismo , Metaloporfirinas/química , Metano/análogos & derivados , Metano/metabolismo , Methanosarcina barkeri/genética , Methanosarcina barkeri/metabolismo , Família Multigênica , Níquel/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Tetrapirróis/química , Uroporfirinas/química , Uroporfirinas/metabolismo
17.
Sci Rep ; 6: 36899, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27845382

RESUMO

Bacterial microcompartments (BMCs) are proteinaceous organelles that are found in a broad range of bacteria and are composed of an outer shell that encases an enzyme cargo representing a specific metabolic process. The outer shell is made from a number of different proteins that form hexameric and pentameric tiles, which interact to allow the formation of a polyhedral edifice. We have previously shown that the Citrobacter freundii BMC associated with 1,2-propanediol utilization can be transferred into Escherichia coli to generate a recombinant BMC and that empty BMCs can be formed from just the shell proteins alone. Herein, a detailed structural and proteomic characterization of the wild type BMC is compared to the recombinant BMC and a number of empty BMC variants by 2D-gel electrophoresis, mass spectrometry, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Specifically, it is shown that the wild type BMC and the recombinant BMC are similar in terms of composition, size, shape and mechanical properties, whereas the empty BMC variants are shown to be smaller, hollow and less malleable.


Assuntos
Citrobacter freundii/metabolismo , Organelas/química , Proteínas de Bactérias/metabolismo , Bioengenharia , Citrobacter freundii/química , Citrobacter freundii/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Propilenoglicol/metabolismo , Proteômica
18.
Angew Chem Int Ed Engl ; 55(37): 11281-6, 2016 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-27355790

RESUMO

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.


Assuntos
Cobamidas/farmacologia , Corrinoides/síntese química , Corrinoides/farmacologia , Desidrogenase do Álcool de Açúcar/antagonistas & inibidores , Citrobacter freundii/enzimologia , Cobamidas/química , Corrinoides/química , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Desidrogenase do Álcool de Açúcar/metabolismo
19.
Sci Rep ; 5: 16943, 2015 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-26616290

RESUMO

CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel ß-sheets have swapped their last ß-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold. The trapped ternary complex with coenzyme and product reveals five conserved basic residues that bind the carboxylates of the tetrapyrrole tightly anchoring the product. A loop, disordered in both the apoenzyme and holoenzyme structures, closes around the product further tightening binding. The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK. The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.


Assuntos
Modelos Moleculares , Oxirredutases/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Uroporfirinas/química , Sítios de Ligação , Catálise , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/metabolismo , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Uroporfirinas/metabolismo
20.
Mol Microbiol ; 93(2): 247-61, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24865947

RESUMO

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.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Desulfovibrio desulfuricans/enzimologia , Desulfovibrio vulgaris/enzimologia , Heme/análogos & derivados , Heme/biossíntese , Methanosarcina barkeri/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biocatálise , Carboxiliases/genética , Carboxiliases/isolamento & purificação , Domínio Catalítico , Desulfovibrio desulfuricans/genética , Desulfovibrio vulgaris/genética , Heme/isolamento & purificação , Heme/metabolismo , Cinética , Methanosarcina barkeri/genética , Oxirredução , Multimerização Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/química
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