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1.
Mol Microbiol ; 118(3): 191-207, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35785499

RESUMO

Some prokaryotes compartmentalize select metabolic capabilities. Salmonella enterica subspecies enterica serovar Typhimurium LT2 (hereafter S. Typhimurium) catabolizes ethanolamine (EA) within a proteinaceous compartment that we refer to as the ethanolamine utilization (Eut) metabolosome. EA catabolism is initiated by the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL), which deaminates EA via an adenosyl radical mechanism to yield acetaldehyde plus ammonia. This adenosyl radical can be quenched, requiring the replacement of AdoCbl by the ATP-dependent EutA reactivase. During growth on ethanolamine, S. Typhimurium synthesizes AdoCbl from cobalamin (Cbl) using the ATP:Co(I)rrinoid adenosyltransferase (ACAT) EutT. It is known that EAL localizes to the metabolosome, however, prior to this work, it was unclear where EutA and EutT localized, and whether they interacted with EAL. Here, we provide evidence that EAL, EutA, and EutT localize to the Eut metabolosome, and that EutA interacts directly with EAL. We did not observe interactions between EutT and EAL nor between EutT and the EutA/EAL complex. However, growth phenotypes of a ΔeutT mutant strain show that EutT is critical for efficient ethanolamine catabolism. This work provides a preliminary understanding of the dynamics of AdoCbl synthesis and its uses within the Eut metabolosome.


Assuntos
Etanolamina Amônia-Liase , Salmonella enterica , Trifosfato de Adenosina/metabolismo , Cobamidas/metabolismo , Etanolamina/metabolismo , Etanolamina Amônia-Liase/genética , Etanolamina Amônia-Liase/metabolismo , Salmonella enterica/genética , Salmonella enterica/metabolismo , Salmonella typhimurium/metabolismo
2.
mBio ; 13(4): e0179322, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35880884

RESUMO

Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.


Assuntos
Acinetobacter baumannii , Etanolamina Amônia-Liase , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Carbono/metabolismo , Cobamidas/metabolismo , Etanolamina/metabolismo , Etanolamina Amônia-Liase/genética , Etanolamina Amônia-Liase/metabolismo , Etanolaminas/metabolismo , Humanos , Salmonella typhimurium/genética
3.
Methods Enzymol ; 669: 151-172, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35644170

RESUMO

Adenosylcobalamin- (AdoCbl) dependent enzyme reactions involved the transfer of hydrogen atoms between the 5'-carbon of the coenzyme and the substrates and products of the reaction. Tritium and deuterium kinetic isotope effect measurements are, therefore, a valuable tool to probe the mechanisms of AdoCbl-dependent enzymes, as they can provide information about the reaction pathway and the rate-determining step. Furthermore, if the intrinsic kinetic isotope effect can be isolated, information on the nature of the transition state associated with hydrogen transfer can be obtained. In this chapter I present methods for the preparation of isotopically-labeled AdoCbl and their use in rapid chemical quench experiments that allow isotope effects on specific steps in the reaction to be isolated. These techniques are illustrated with examples from my laboratory's studies on the AdoCbl dependent enzyme, glutamate mutase.


Assuntos
Cobamidas , Isótopos , Cobamidas/metabolismo , Hidrogênio/metabolismo , Cinética
4.
Methods Enzymol ; 668: 181-242, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589194

RESUMO

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.


Assuntos
Etanolamina Amônia-Liase , Animais , Cobamidas/química , Cobamidas/metabolismo , Etanolamina Amônia-Liase/química , Etanolamina Amônia-Liase/metabolismo , Glicerol , Hidroliases , Fosfotreonina/análogos & derivados
5.
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
6.
Methods Enzymol ; 668: 349-372, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589201

RESUMO

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Cobamidas/química , Cobamidas/genética , Cobamidas/metabolismo , DNA/metabolismo , Fosfotreonina/análogos & derivados , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , Vitamina B 12/metabolismo
7.
Methods Enzymol ; 668: 61-85, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589202

RESUMO

Cobamides are a family of enzyme cofactors that are required by organisms in all domains of life. Over a dozen cobamides exist in nature although only cobalamin (vitamin B12), the cobamide required by humans, has been studied extensively. Cobamides are exclusively produced by a subset of prokaryotes. Importantly, the bacteria and archaea that synthesize cobamides de novo typically produce a single type of cobamide, and furthermore, organisms that use cobamides are selective for certain cobamides. Therefore, a detailed understanding of the cobamide-dependent metabolism of an organism or microbial community of interest requires experiments performed with a variety of cobamides. A notable challenge is that cobalamin is the only cobamide that is commercially available at present. In this chapter, we describe methods to extract, purify, and quantify various cobamides from bacteria for use in laboratory experiments.


Assuntos
Cobamidas , Vitamina B 12 , Bactérias/metabolismo , Cobamidas/metabolismo , Coenzimas , Humanos , Vitamina B 12/metabolismo , Vitaminas
8.
Methods Enzymol ; 668: 109-123, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589190

RESUMO

Cobamides are essential for the performance of a variety of reactions such methyl transfers, carbon skeleton rearrangements, and eliminations in both prokaryotes and eukaryotes. However, cobamide biosynthesis is limited to a subset of bacteria and archaea. The biosynthesis pathway culminates with the activation and attachment of a lower ligand to the corrin ring; this branch of the pathway is known as nucleotide loop assembly (NLA) pathway. The cobamide synthase (CobS) enzyme is the penultimate step in NLA pathway, and catalyzes the attachment of an α-ribotide to the activated corrin ring. While other NLA enzymes have been well-studied, studies of CobS have proven difficult to date. CobS is an integral membrane protein, and limitations have been largely due to difficulties in protein purification. Here we provide a method to purify CobS, reconstitute protein in proteoliposomes, and assay for its activity.


Assuntos
Cobamidas , Lipossomos , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Cobamidas/metabolismo
9.
Methods Enzymol ; 668: 125-136, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589191

RESUMO

Cobamides (Cbas) are the largest coenzymes known and are used by cells in all domains of life. These molecules are characterized by a central cobalt-containing tetrapyrrole ring with two opposing axial ligands on the α and ß faces of the ring. All biologically active forms of Cbas have a 5'-deoxyadenosyl group as the upper (Coß) ligand that is covalently attached to the cobalt ion of the ring. In contrast, the lower ligand is a nucleobase of diverse chemical structure; however, nucleobases are usually derivatives of benzimidazole or purine. Phenol and p-cresol can also serve as the nucleobase, but they cannot form a coordination bond with the cobalt ion of the ring because they lack a free pair of electrons. The Cba incorporating 5,6-dimethylbenzimidazole (DMB) is known as cobalamin (Cbl), and the coenzymic form of cobalamin is known as adenosylcobalamin (AdoCbl). A common vitamer of cobalamin has a cyano group as the upper ligand. This vitamer is known as cyanocobalamin (CNCbl), which is commercially marketed as vitamin B12. Here, we describe a combination of chemical hydrolysis of cobalamin with the enzymatic dephosphorylation of the resulting α-R-3'-phosphate to yield α-R, which we enzymically convert to the pathway intermediate α-R-5'-phosphate (α-RP). The methods describe herein can be readily scaled up to generate large amounts of α-RP.


Assuntos
Fosfatos , Vitamina B 12 , Cobalto/química , Cobamidas/química , Cobamidas/metabolismo , Coenzimas , Ligantes , Ribonucleosídeos , Vitamina B 12/metabolismo , Vitaminas
10.
Vitam Horm ; 119: 43-63, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35337629

RESUMO

Cobamides are a family of structurally-diverse cofactors which includes vitamin B12 and over a dozen natural analogs. Within the nucleotide loop structure, cobamide analogs have variable lower ligands that fall into three categories: benzimidazoles, purines, and phenols. The range of cobamide analogs that can be utilized by an organism is dependent on the specificity of its cobamide-dependent enzymes, and most bacteria are able to utilize multiple analogs but not all. Some bacteria have pathways for cobamide remodeling, a process in which imported cobamides are converted into compatible analogs. Here we discuss cobamide analog diversity and three pathways for cobamide remodeling, mediated by amidohydrolase CbiZ, phosphodiesterase CbiR, and some homologs of cobamide synthase CobS. Remodeling proteins exhibit varying degrees of specificity for cobamide substrates, reflecting different strategies to ensure that imported cobamides can be utilized.


Assuntos
Cobamidas , Vitamina B 12 , Cobamidas/química , Cobamidas/metabolismo , Humanos , Ligantes , Vitamina B 12/metabolismo
11.
Enzyme Microb Technol ; 157: 110021, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35231673

RESUMO

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.


Assuntos
Propanodiol Desidratase , Cobamidas/metabolismo , Glicerol/metabolismo , Hidroliases/genética , Hidroliases/metabolismo , Klebsiella pneumoniae/genética , Óperon , Propanodiol Desidratase/genética , Propanodiol Desidratase/metabolismo
12.
mBio ; 11(6)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293380

RESUMO

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling-the removal and replacement of the lower ligand-despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobamidas/metabolismo , Akkermansia/enzimologia , Akkermansia/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cromatografia Líquida de Alta Pressão , Cobamidas/química , Humanos , Hidrólise , Estrutura Molecular , Vitamina B 12/química
13.
Proc Natl Acad Sci U S A ; 117(48): 30412-30422, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199623

RESUMO

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt-carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Cobamidas/química , Cobamidas/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cinética , Modelos Biológicos , Conformação Molecular , Complexos Multiproteicos , Ligação Proteica , Relação Estrutura-Atividade
14.
FEMS Microbiol Lett ; 367(20)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33152082

RESUMO

Microalgae are not able to produce cobamides (Cbas, B12 vitamers) de novo. Hence, the production of catalytically active Cba-containing methionine synthase (MetH), which is present in selected representatives, is dependent on the availability of exogenous B12 vitamers. Preferences in the utilization of exogenous Cbas equipped with either adenine or 5,6-dimethylbenzimidazole as lower base have been reported for some microalgae. Here, we investigated the utilization of norcobamides (NorCbas) for growth by the Cba-dependent Chlamydomonas reinhardtii mutant strain (ΔmetE). The growth yields in the presence of NorCbas were lower in comparison to those achieved with Cbas. NorCbas lack a methyl group in the linker moiety of the nucleotide loop. C. reinhardtii was also tested for the remodeling of NorCbas (e.g. adeninyl-norcobamide) in the presence of different benzimidazoles. Extraction of the NorCbas from C. reinhardtii, their purification, and identification confirmed the exchange of the lower base of the vitamers. However, the linker moiety of the NorCbas nucleotide loop was not exchanged. This observation strongly indicates the presence of an alternative mode of Cba deconstruction in C. reinhardtii that differs from the amidohydrolase (CbiZ)-dependent pathway described in Cba-remodeling bacteria and archaea.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Cobamidas/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cobamidas/química , Água Doce
15.
Science ; 369(6499)2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631870

RESUMO

Microbial communities are essential to fundamental processes on Earth. Underlying the compositions and functions of these communities are nutritional interdependencies among individual species. One class of nutrients, cobamides (the family of enzyme cofactors that includes vitamin B12), is widely used for a variety of microbial metabolic functions, but these structurally diverse cofactors are synthesized by only a subset of bacteria and archaea. Advances at different scales of study-from individual isolates, to synthetic consortia, to complex communities-have led to an improved understanding of cobamide sharing. Here, we discuss how cobamides affect microbes at each of these three scales and how integrating different approaches leads to a more complete understanding of microbial interactions.


Assuntos
Cobamidas/metabolismo , Meio Ambiente , Interações Microbianas , Microbiota , Complexo Vitamínico B/metabolismo , Animais , Archaea/metabolismo , Bactérias/metabolismo , Cobamidas/química , Planeta Terra , Eucariotos/metabolismo , Modelos Biológicos , Complexo Vitamínico B/química
16.
Biochemistry ; 59(10): 1124-1136, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32125848

RESUMO

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.


Assuntos
Corrinoides/metabolismo , Listeria monocytogenes/enzimologia , Aciltransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/ultraestrutura , Alquil e Aril Transferases/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Catálise , Domínio Catalítico , Cobalto/química , Cobamidas/metabolismo , Cinética , Lactobacillus reuteri/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutação , Transferases/metabolismo
17.
Curr Biol ; 30(2): R55-R56, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31962073

RESUMO

Vitamin B12 is the only known essential human micronutrient made exclusively by prokaryotes. Kennedy and Taga introduce us to the world of cobamides-those cobalt-containing compounds, like B12, that appear to be the proprietary domain of our microbial partners.


Assuntos
Anti-Infecciosos , Cobamidas , Complexo Vitamínico B , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Cobamidas/química , Cobamidas/metabolismo , Cobamidas/farmacologia , Humanos , Complexo Vitamínico B/química , Complexo Vitamínico B/metabolismo , Complexo Vitamínico B/farmacologia
19.
J Bacteriol ; 202(2)2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31685533

RESUMO

Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.


Assuntos
Clostridioides difficile/metabolismo , Cobamidas/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Ácido Aminolevulínico/metabolismo , Ribonucleotídeo Redutases/metabolismo , Vitamina B 12/metabolismo
20.
Mol Microbiol ; 113(1): 89-102, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31609521

RESUMO

Cobamides are a group of compounds including vitamin B12 that can vary at the lower base position of the nucleotide loop. They are synthesized de novo by only a subset of prokaryotes, but some organisms encode partial biosynthesis pathways for converting one variant to another (remodeling) or completing biosynthesis from an intermediate (corrinoid salvaging). Here, we explore the cobamide specificity in Vibrio cholerae through examination of three natural variants representing major cobamide groups: commercially available cobalamin, and isolated pseudocobalamin and p-cresolylcobamide. We show that BtuB, the outer membrane corrinoid transporter, mediates the uptake of all three variants and the intermediate cobinamide. Our previous work suggested that V. cholerae could convert pseudocobalamin produced by cyanobacteria into cobalamin. In this work, cobamide specificity in V. cholerae is demonstrated by remodeling of pseudocobalamin and salvaging of cobinamide to produce cobalamin. Cobamide remodeling in V. cholerae is distinct from the canonical pathway requiring amidohydrolase CbiZ, and heterologous expression of V. cholerae CobS was sufficient for remodeling. Furthermore, function of V. cholerae cobamide-dependent methionine synthase MetH was robustly supported by cobalamin and p-cresolylcobamide, but not pseudocobalamin. Notably, the inability of V. cholerae to produce and utilize pseudocobalamin contrasts with enteric bacteria like Salmonella.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Cobamidas/metabolismo , Vibrio cholerae/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Transporte Biológico
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