RESUMO
Carbon and chlorine isotope effects for biotransformation of chloroform by different microbes show significant variability. Reductive dehalogenases (RDase) enzymes contain different cobamides, affecting substrate preferences, growth yields, and dechlorination rates and extent. We investigate the role of cobamide type on carbon and chlorine isotopic signals observed during reductive dechlorination of chloroform by the RDase CfrA. Microcosm experiments with two subcultures of a Dehalobacter-containing culture expressing CfrA-one with exogenous cobamide (Vitamin B12, B12+) and one without (to drive native cobamide production)-resulted in a markedly smaller carbon isotope enrichment factor (εC, bulk) for B12- (-22.1 ± 1.9) compared to B12+ (-26.8 ± 3.2). Both cultures exhibited significant chlorine isotope fractionation, and although a lower εCl, bulk was observed for B12- (-6.17 ± 0.72) compared to B12+ (-6.86 ± 0.77) cultures, these values are not statistically different. Importantly, dual-isotope plots produced identical slopes of ΛCl/C (ΛCl/C, B12+ = 3.41 ± 0.15, ΛCl/C, B12- = 3.39 ± 0.15), suggesting the same reaction mechanism is involved in both experiments, independent of the lower cobamide bases. A nonisotopically fractionating masking effect may explain the smaller fractionations observed for the B12- containing culture.
Assuntos
Biotransformação , Clorofórmio , Vitamina B 12 , Clorofórmio/metabolismo , Vitamina B 12/metabolismo , Cloro/metabolismo , Isótopos de Carbono/metabolismo , Cobamidas/metabolismoRESUMO
3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical centre compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.
Assuntos
Acetaldeído , Cobamidas , Propanodiol Desidratase , Acetaldeído/metabolismo , Acetaldeído/química , Propanodiol Desidratase/metabolismo , Propanodiol Desidratase/química , Cobamidas/metabolismo , Cobamidas/química , Fluoretos/metabolismo , Fluoretos/química , Propilenoglicóis/metabolismo , Propilenoglicóis/químicaRESUMO
The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.
Assuntos
Corrinoides , Riboswitch , Vitamina B 12 , Vitamina B 12/química , Vitamina B 12/metabolismo , Corrinoides/química , Corrinoides/metabolismo , Ligantes , Ligação de Hidrogênio , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Cobamidas/química , Cobamidas/metabolismo , Sítios de Ligação , Proteínas de Membrana TransportadorasRESUMO
Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.
Assuntos
Alteromonadaceae , Ligantes , Rhodobacteraceae , Vitamina B 12 , Oceano Atlântico , Técnicas de Cocultura , Interações Microbianas , Prófagos/genética , Prófagos/crescimento & desenvolvimento , Prófagos/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/química , Vitamina B 12/metabolismo , Alteromonadaceae/crescimento & desenvolvimento , Alteromonadaceae/metabolismo , Rhodobacteraceae/citologia , Rhodobacteraceae/metabolismo , Rhodobacteraceae/virologia , Ribonucleosídeos/metabolismo , Cobamidas/metabolismo , EcossistemaRESUMO
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
Assuntos
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Mensageiro , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Cobamidas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Helicases/genética , RNA Helicases/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas da Membrana Bacteriana Externa , Polirribonucleotídeo Nucleotidiltransferase , Proteínas de Membrana TransportadorasRESUMO
BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the ß-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the ß-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 µM), followed by the formation of a covalent bond (rate constant of 0.163 s-1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.
Assuntos
Proteínas de Bactérias , Modelos Moleculares , Ligação Proteica , Vitamina B 12 , Vitamina B 12/metabolismo , Vitamina B 12/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Sítios de Ligação , Cobalto/química , Cobalto/metabolismo , Cobamidas/metabolismo , Cobamidas/química , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Histidina/metabolismo , Histidina/químicaRESUMO
Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathwayâisomerization of ß-l-lysine to erythro-3,5-diaminohexanoateâis catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.
Assuntos
Transferases Intramoleculares , Lisina , Lisina/metabolismo , Transferases Intramoleculares/metabolismo , Cobamidas/metabolismo , Trifosfato de AdenosinaRESUMO
G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.
Assuntos
Cobamidas , Metilmalonil-CoA Mutase , Modelos Moleculares , Chaperonas Moleculares , Cobamidas/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Isomerases/química , Isomerases/metabolismo , Metilmalonil-CoA Mutase/química , Metilmalonil-CoA Mutase/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Cupriavidus/química , Cupriavidus/enzimologia , Estrutura Quaternária de Proteína , Domínio Catalítico , Coenzimas/metabolismoRESUMO
Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation.
Assuntos
Proteínas de Bactérias , Técnicas Biossensoriais , Humanos , Proteínas de Bactérias/química , Cobamidas/química , Cobamidas/metabolismo , Vitamina B 12/metabolismo , Bactérias/metabolismoRESUMO
Vitamin B12 is an essential biomolecule that assists in the catalysis of methyl transfer and radical-based reactions in cellular metabolism. The structure of B12 is characterized by a tetrapyrrolic corrin ring with a central cobalt ion coordinated with an upper ligand, and a lower ligand anchored via a nucleotide loop. Multiple methyl groups decorate B12, and their presence (or absence) have structural and functional consequences. In this minireview, we focus on the methyl groups that distinguish vitamin B12 from other tetrapyrrolic biomolecules and from its own naturally occurring analogues called cobamides. We draw information from recent advances in the field to understand the origins of these methyl groups and the enzymes that incorporate them, and discuss their biological significance.
Assuntos
Cobamidas , Vitamina B 12 , Vitamina B 12/metabolismo , Ligantes , Cobamidas/química , Cobamidas/metabolismo , Catálise , VitaminasRESUMO
Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5'-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5'-phosphate (α-RP) or α-adenylyl-5'-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT.
Assuntos
Cianobactérias , Euryarchaeota , Monofosfato de Adenosina , Archaea/metabolismo , Ácido Aspártico , Cobamidas/metabolismo , Cristalografia por Raios X , Cianobactérias/metabolismo , Euryarchaeota/metabolismo , Glutamatos , Ligantes , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Fosfatos/metabolismoRESUMO
The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.
Assuntos
Etanolamina Amônia-Liase , Propanodiol Desidratase , Etanolamina Amônia-Liase/química , Etanolamina Amônia-Liase/metabolismo , Propanodiol Desidratase/química , Propanodiol Desidratase/metabolismo , Cobamidas/química , Cobamidas/metabolismo , CinéticaRESUMO
Vitamin B12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.
Assuntos
Cobamidas , Vitamina B 12 , Animais , Bactérias/metabolismo , Cobamidas/metabolismo , Mamíferos/metabolismo , Vitamina B 12/metabolismo , Corrinoides/química , Corrinoides/metabolismoRESUMO
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/metabolismoRESUMO
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/genéticaRESUMO
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 , CinéticaRESUMO
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 , VitaminasRESUMO
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/metabolismoRESUMO
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 & derivadosRESUMO
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.