RESUMO
Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.
Assuntos
Mimetismo Molecular , Vitamina B 12 , Humanos , Oxirredução , Difosfato de Adenosina/metabolismo , Vitamina B 12/metabolismo , Metilmalonil-CoA Mutase/química , Metilmalonil-CoA Mutase/metabolismoRESUMO
In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.
Assuntos
Vitamina B 12 , Vitamina B 12/metabolismo , Vitamina B 12/química , Vitamina B 12/análogos & derivados , Humanos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Metilmalonil-CoA Mutase/química , Ródio/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Mimetismo Molecular , Modelos Moleculares , Alquil e Aril TransferasesRESUMO
Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (â¼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.
Assuntos
Cistationina beta-Sintase , Mutação , Humanos , Regulação Alostérica/genética , Cristalografia por Raios X , Cistationina beta-Sintase/química , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Homocisteína/metabolismo , Homocistinúria/enzimologia , Homocistinúria/genética , Cinética , S-Adenosilmetionina/metabolismo , Conformação Proteica , Domínio CatalíticoRESUMO
Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in nature. Cobalamin (or vitamin B12) is an essential cobalt-containing organometallic cofactor in mammals and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination for cofactor off-loading onto methionine synthase. Cys-261 on MMADHC serves as the ß-axial ligand to cobalamin. Complex formation between MMADHC and methionine synthase is signaled by loss of the lower axial nitrogen ligand, leading to five-coordinate thiolato-cobalamin. Nucleophilic displacement by the vicinal thiolate, Cys-262, completes cofactor transfer to methionine synthase and release of a cysteine disulfide-containing MMADHC. The physiological relevance of this mechanism is supported by clinical variants of MMADHC, which impair cofactor binding and off-loading, explaining the molecular basis of the associated homocystinuria.
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-AtividadeRESUMO
Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5'-deoxyadenosylcobalamin (AdoCbl) or coenzyme B12. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase KM(ATP). 31P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the kcat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.
Assuntos
Trifosfato de Adenosina/metabolismo , Alquil e Aril Transferases/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Alquil e Aril Transferases/química , Catálise , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinética , Ligação ProteicaRESUMO
In humans, cobalamin or vitamin B12 is delivered to two target enzymes via a complex intracellular trafficking pathway comprising transporters and chaperones. CblC (or MMACHC) is a processing chaperone that catalyzes an early step in this trafficking pathway. CblC removes the upper axial ligand of cobalamin derivatives, forming an intermediate in the pathway that is subsequently converted to the active cofactor derivatives. Mutations in the cblC gene lead to methylmalonic aciduria and homocystinuria. Here, we report that nitrosylcobalamin (NOCbl), which was developed as an antiproliferative reagent, and is purported to cause cell death by virtue of releasing nitric oxide, is highly unstable in air and is rapidly oxidized to nitrocobalamin (NO2Cbl). We demonstrate that CblC catalyzes the GSH-dependent denitration of NO2Cbl forming 5-coordinate cob(II)alamin, which had one of two fates. It could be oxidized to aquo-cob(III)alamin or enter a futile thiol oxidase cycle forming GSH disulfide. Arg-161 in the active site of CblC suppressed the NO2Cbl-dependent thiol oxidase activity, whereas the disease-associated R161G variant stabilized cob(II)alamin and promoted futile cycling. We also report that CblC exhibits nitrite reductase activity, converting cob(I)alamin and nitrite to NOCbl. Finally, the denitration activity of CblC supported cell proliferation in the presence of NO2Cbl, which can serve as a cobalamin source. The newly described nitrite reductase and denitration activities of CblC extend its catalytic versatility, adding to its known decyanation and dealkylation activities. In summary, upon exposure to air, NOCbl is rapidly converted to NO2Cbl, which is a substrate for the B12 trafficking enzyme CblC.
Assuntos
Nitrito Redutases , Oxirredutases , Vitamina B 12/análogos & derivados , Transporte Biológico Ativo , Catálise , Células HT29 , Humanos , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMO
The pathways for biosynthesis of glycogen in bacteria and starch in plants are evolutionarily and biochemically related. They are regulated primarily by ADP-glucose pyrophosphorylase, which evolved to satisfy metabolic requirements of a particular organism. Despite the importance of these two pathways, little is known about the mechanism that controls pyrophosphorylase activity or the location of its allosteric sites. Here, we report pyruvate-bound crystal structures of ADP-glucose pyrophosphorylase from the bacterium Agrobacterium tumefaciens, identifying a previously elusive activator site for the enzyme. We found that the tetrameric enzyme binds two molecules of pyruvate in a planar conformation. Each binding site is located in a crevice between the C-terminal domains of two subunits where they stack via a distinct ß-helix region. Pyruvate interacts with the side chain of Lys-43 and with the peptide backbone of Ser-328 and Gly-329 from both subunits. These structural insights led to the design of two variants with altered regulatory properties. In one variant (K43A), the allosteric effect was absent, whereas in the other (G329D), the introduced Asp mimicked the presence of pyruvate. The latter generated an enzyme that was preactivated and insensitive to further activation by pyruvate. Our study furnishes a deeper understanding of how glycogen biosynthesis is regulated in bacteria and the mechanism by which transgenic plants increased their starch production. These insights will facilitate rational approaches to enzyme engineering for starch production in crops of agricultural interest and will promote further study of allosteric signal transmission and molecular evolution in this important enzyme family.
Assuntos
Agrobacterium tumefaciens/enzimologia , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Piruvatos/metabolismo , Sítios de Ligação , Glucose-1-Fosfato Adenililtransferase/genética , Glicogênio/biossíntese , Glicogênio/química , Modelos Moleculares , Estrutura MolecularRESUMO
The CblC and CblD chaperones are involved in early steps in the cobalamin trafficking pathway. Cobalamin derivatives entering the cytoplasm are converted by CblC to a common cob(II)alamin intermediate via glutathione-dependent alkyltransferase or reductive elimination activities. Cob(II)alamin is subsequently converted to one of two biologically active alkylcobalamins by downstream chaperones. The function of CblD has been elusive although it is known to form a complex with CblC under certain conditions. Here, we report that CblD provides a sulfur ligand to cob(II)alamin bound to CblC, forming an interprotein coordination complex that rapidly oxidizes to thiolato-cob(III)alamin. Cysteine scanning mutagenesis and EPR spectroscopy identified Cys-261 on CblD as the sulfur donor. The unusual interprotein Co-S bond was characterized by X-ray absorption spectroscopy and visualized in the crystal structure of the human CblD thiolato-cob(III)alamin complex. Our study provides insights into how cobalamin coordination chemistry could be utilized for cofactor translocation in the trafficking pathway.
Assuntos
Cobalto/metabolismo , Chaperonas Moleculares/metabolismo , Enxofre/metabolismo , Vitamina B 12/metabolismo , Cobalto/química , Modelos Moleculares , Chaperonas Moleculares/química , Enxofre/química , Vitamina B 12/químicaRESUMO
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Inhibition of GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, has been established as a possible strategy for the treatment of substance abuse. The raised GABA levels that occur as a consequence of this inhibition have been found to antagonize the rapid release of dopamine in the ventral striatum (nucleus accumbens) that follows an acute challenge by an addictive substance. In addition, increased GABA levels are also known to elicit an anticonvulsant effect in patients with epilepsy. We previously designed the mechanism-based inactivator (1S,3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (2), now called CPP-115, that is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved drug that is an inactivator of GABA-AT. CPP-115 was found to have high therapeutic potential for the treatment of cocaine addiction and for a variety of epilepsies, has successfully completed a Phase I safety clinical trial, and was found to be effective in the treatment of infantile spasms (West syndrome). Herein we report the design, using molecular dynamics simulations, synthesis, and biological evaluation of a new mechanism-based inactivator, (S)-3-amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (5), which was found to be almost 10 times more efficient as an inactivator of GABA-AT than CPP-115. We also present the unexpected crystal structure of 5 bound to GABA-AT, as well as computational analyses used to assist the structure elucidation process. Furthermore, 5 was found to have favorable pharmacokinetic properties and low off-target activities. In vivo studies in freely moving rats showed that 5 was dramatically superior to CPP-115 in suppressing the release of dopamine in the corpus striatum, which occurs subsequent to either an acute cocaine or nicotine challenge. Compound 5 also attenuated increased metabolic demands (neuronal glucose metabolism) in the hippocampus, a brain region that encodes spatial information concerning the environment in which an animal receives a reinforcing or aversive drug. This multidisciplinary computational design to preclinical efficacy approach should be applicable to the design and improvement of mechanism-based inhibitors of other enzymes whose crystal structures and inactivation mechanisms are known.
Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Prolina/análogos & derivados , 4-Aminobutirato Transaminase/química , 4-Aminobutirato Transaminase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Dopamina/metabolismo , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacocinética , Antagonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacocinética , Glucose/metabolismo , Humanos , Masculino , Modelos Moleculares , Prolina/química , Prolina/farmacocinética , Prolina/farmacologia , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismoRESUMO
Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.
Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Aspartato Aminotransferases/antagonistas & inibidores , Cicloleucina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Fosfato de Piridoxal/metabolismo , 4-Aminobutirato Transaminase/química , 4-Aminobutirato Transaminase/metabolismo , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cicloleucina/química , Cicloleucina/metabolismo , Cicloleucina/farmacologia , Bases de Dados de Compostos Químicos , Bases de Dados de Proteínas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ligantes , Conformação Molecular , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Conformação Proteica , Fosfato de Piridoxal/química , Piridoxamina/química , Piridoxamina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 10(6) M(-1) s(-1) for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 µM for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed.
Assuntos
Hidrolases de Éster Carboxílico/química , Chryseobacterium/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Chryseobacterium/química , Chryseobacterium/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Percepção de Quorum , Especificidade por SubstratoRESUMO
G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. The G-protein, MMAA, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B12-dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the complex assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nano-assembly, which reveals a dramatic 180° rotation of the B12 domain, exposing it to solvent. The complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the MMAA-MMUT interfaces we identify here.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Transferases Intramoleculares , Humanos , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Mutação , Erros Inatos do Metabolismo dos Aminoácidos/genética , Proteínas de Ligação ao GTP/genética , GTP Fosfo-Hidrolases/metabolismo , Transferases Intramoleculares/genéticaRESUMO
G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. MMAA, a G-protein motor, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B 12 -dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the motor assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nanomotor assembly, which reveals a dramatic 180° rotation of the B 12 domain, exposing it to solvent. The nanomotor complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the newly identified MMAA-MMUT interfaces.
RESUMO
Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in Nature. Cobalamin (or vitamin B 12 ) is an essential cobalt-containing organometallic cofactor in mammals, and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination, for cofactor off-loading onto methionine synthase. Cys-261 on MMADHC serves as the ß-axial ligand to cobalamin. Complex formation between MMADHC and methionine synthase is signaled by loss of the lower axial nitrogen ligand, leading to five-coordinate thiolato-cobalamin. Nucleophilic displacement by the vicinal thiolate, Cys-262, completes cofactor transfer to methionine synthase and release of a cysteine disulfide-containing MMADHC. The physiological relevance of this mechanism is supported by clinical variants of MMADHC, which impair cofactor binding and off-loading, explaining the molecular basis of the associated homocystinuria.
RESUMO
Humans have only two known cobalamin or B12-dependent enzymes: cytoplasmic methionine synthase and mitochondrial methylmalonyl-CoA mutase. A complex intracellular B12 trafficking pathway, comprising a multitude of chaperones, process and deliver cobalamin to the two target enzymes. Methionine synthase catalyzes the transfer of a methyl group from N5-methytetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Cobalamin serves as an intermediate methyl group carrier and cycles between methylcobalamin and cob(I)alamin. Methylmalonyl-CoA mutase uses the 5'-deoxyadenosylcobalamin form of the cofactor and catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. Two chaperones, CblA (or MMAA) and CblB (or MMAB, also known as adenosyltransferase), serve the mutase and ensure that the fidelity of the cofactor loading and unloading processes is maintained. This chapter focuses on assays for purifying and measuring the activities of methionine synthase and methylmalonyl-CoA mutase.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase , Metilmalonil-CoA Mutase , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Humanos , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Vitamina B 12/metabolismoRESUMO
An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt(II) AiiA with an inhibition constant of K i = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate-enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process.
RESUMO
Bacterial ADP-glucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. Pyruvate (Pyr) and fructose 6-phosphate (Fru6P) activate the enzyme from Agrobacterium tumefaciens by increasing Vmax about 10- and 20-fold, respectively. Here, we studied the combined effect of both metabolites on the enzyme activation. Our results support a model in which there is a synergistic binding of these two activators to two distinct sites and that each activator leads the enzyme to distinct active forms with different properties. In presence of both activators, Pyr had a catalytically dominant effect over Fru6P determining the active conformational state. By mutagenesis we obtained enzyme variants still sensitive to Pyr activation, but in which the allosteric signal by Fru6P was disrupted. This indicated that the activation mechanism for each effector was not the same. The ability for this enzyme to have more than one allosteric activator site, active forms, and allosteric signaling mechanisms is critical to expand the evolvability of its regulation. These synergistic interactions between allosteric activators may represent a feature in other allosteric enzymes.