RESUMO
Local reactivity descriptors such as atom-condensed Fukui functions are promising computational tools to study chemical reactivity at specific sites within a molecule. Their applications have been mainly focused on isolated molecules in their most stable conformation without considering the effects of the surroundings. Here we propose to combine quantum mechanics/molecular mechanics Born-Oppenheimer molecular dynamics simulations to obtain the microstates (configurations) of a molecular system using different representations of the molecular environment and calculate Boltzmann-weighted atom-condensed local reactivity descriptors based on conceptual density functional theory. Our approach takes the conformational fluctuations of the molecular system and the polarization of its electron density by the environment into account, allowing us to analyze the effect of the molecular environment on reactivity. In this contribution, we apply the method mentioned above to the catalytic fixation of carbon dioxide by crotonyl-CoA carboxylase/reductase and study if the enzyme alters the reactivity of its substrate compared with an aqueous solution. Our main result is that the protein environment activates the substrate by the elimination of solute-solvent hydrogen bonds from aqueous solution in the two elementary steps of the reaction mechanism: the nucleophilic attack of a hydride anion from NADPH on the α,ß-unsaturated thioester and the electrophilic attack of carbon dioxide on the formed enolate species.
Assuntos
Dióxido de Carbono/química , Carbono-Carbono Ligases/química , Acil Coenzima A/química , Teoria da Densidade Funcional , Ligação de Hidrogênio , Modelos Químicos , Simulação de Dinâmica Molecular , NADP/químicaRESUMO
Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace_4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE_4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace_4237 is essential for S. erythraea survival as determined by the construction of a sace_4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.
Assuntos
Carbono-Carbono Ligases/metabolismo , Ácidos Graxos/biossíntese , Malonil Coenzima A/metabolismo , Saccharopolyspora/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/genética , Filogenia , Domínios Proteicos , Saccharopolyspora/genética , Saccharopolyspora/crescimento & desenvolvimentoRESUMO
Biotin is a water-soluble vitamin that belongs to the vitamin B complex and which is an essential nutrient of all living organisms from bacteria to man. In eukaryotic cells biotin functions as a prosthetic group of enzymes, collectively known as biotin-dependent carboxylases that catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Enzyme-bound biotin acts as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In recent years, evidence has mounted that biotin also regulates gene expression through a mechanism beyond its role as a prosthetic group of carboxylases. These activities may offer a mechanistic background to a developing literature on the action of biotin in neurological disorders. This review summarizes the role of biotin in activating carboxylases and proposed mechanisms associated with a role in gene expression and in ameliorating neurological disease.
Assuntos
Biotina/metabolismo , Deficiência de Biotinidase/enzimologia , Biotinidase/metabolismo , Carbono-Carbono Ligases/metabolismo , Aminoácidos/metabolismo , Biotina/deficiência , Deficiência de Biotinidase/genética , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Deficiência Múltipla de Carboxilase/genética , Deficiência Múltipla de Carboxilase/metabolismoRESUMO
Citrus canker is a disease caused by the phytopathogen Xanthomonas citri subsp. citri (Xcc), bacterium which is unable to survive out of the host for extended periods of time. Once established inside the plant, the pathogen must compete for resources and evade the defenses of the host cell. However, a number of aspects of Xcc metabolic and nutritional state, during the epiphytic stage and at different phases of infection, are poorly characterized. The 3-methylcrotonyl-CoA carboxylase complex (MCC) is an essential enzyme for the catabolism of the branched-chain amino acid leucine, which prevents the accumulation of toxic intermediaries, facilitates the generation of branched chain fatty acids and/or provides energy to the cell. The MCC complexes belong to a group of acyl-CoA carboxylases (ACCase) enzymes dependent of biotin. In this work, we have identified two ORFs (XAC0263 and XAC0264) encoding for the α and ß subunits of an acyl-CoA carboxylase complex from Xanthomonas and demonstrated that this enzyme has MCC activity both in vitro and in vivo. We also found that this MCC complex is conserved in a group of pathogenic gram negative bacteria. The generation and analysis of an Xcc mutant strain deficient in MCC showed less canker lesions in the interaction with the host plant, suggesting that the expression of these proteins is necessary for Xcc fitness during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Carbono Ligases/metabolismo , Citrus/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Carbono-Carbono Ligases/genética , Cinética , Leucina/metabolismo , Mutagênese , Fases de Leitura Aberta/genética , Estabilidade Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Xanthomonas/crescimento & desenvolvimento , Xanthomonas/fisiologiaRESUMO
Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Carbono Ligases/metabolismo , Mycobacterium tuberculosis/metabolismo , Policetídeo Sintases/metabolismo , Subunidades Proteicas/metabolismo , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Carbono-Carbono Ligases/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Malonil Coenzima A/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Policetídeo Sintases/genética , Engenharia de Proteínas , Subunidades Proteicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.
Assuntos
Carbono-Carbono Ligases/genética , Parede Celular/genética , Lipídeos/biossíntese , Mycobacterium smegmatis/genética , Acetilcoenzima A , Acil Coenzima A , Sequência de Aminoácidos , Parede Celular/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Lipogênese , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismoRESUMO
Isolated 3-methylcrotonyl-CoA carboxylase deficiency (3MCCD) is an autosomal recessive disorder of leucine metabolism biochemically characterized by accumulation of 3-methylcrotonylglycine (3MCG), 3-methylcrotonic acid (3MCA) and 3-hydroxyisovaleric acid. A considerable number of affected individuals present neurological symptoms with or without precedent crises of metabolic decompensation and brain abnormalities whose pathogenesis is poorly known. We investigated the in vitro effects of 3MCG and 3MCA on important parameters of oxidative stress in cerebral cortex of young rats. 3MCG and 3MCA significantly increased TBA-RS and carbonyl formation, indicating that these compounds provoke lipid and protein oxidation, respectively. In contrast, nitric oxide production was not affected by 3MCG and 3MCA. Furthermore, 3MCG- and 3MCA-induced elevation of TBA-RS values was fully prevented by melatonin, trolox and reduced glutathione, but not by the nitric oxide inhibitor N(ω)-nitro-L-arginine methyl ester or the combination of catalase plus superoxide dismutase, indicating that reactive oxygen species were involved in the oxidative damage caused by these compounds. We also found that the activity of the antioxidant enzymes glutathione peroxidase, catalase, superoxide dismutase and glutathione reductase were not altered in vitro by 3MCG and 3MCA. It is therefore presumed that alterations of the cellular redox homeostasis caused by the major metabolites accumulating in 3MCCD may potentially be involved in the pathophysiology of the neurological dysfunction and structural brain alterations found in patients affected by this disorder.
Assuntos
Química Encefálica/fisiologia , Carbono-Carbono Ligases/deficiência , Córtex Cerebral/metabolismo , Estresse Oxidativo/fisiologia , Fatores Etários , Animais , Córtex Cerebral/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/enzimologia , Distúrbios Congênitos do Ciclo da Ureia/fisiopatologiaRESUMO
Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and ß), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αß)(4) complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30º C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K(m) for its substrate and V(max) of 168 µM and 430 nmoles mg(-1)min(-1), respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.
Assuntos
Carbono-Carbono Ligases/genética , Carbono-Carbono Ligases/isolamento & purificação , Expressão Gênica , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Biotecnologia/métodos , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/metabolismo , Cromatografia de Afinidade , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Plasmídeos , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , Fatores de TempoRESUMO
A neonate who received vitamin K (VK) supplementation then developed severe late-onset bleeding with abnormal prothrombin time and activated partial thromboplastine time. The bleeding was corrected after intravenous VK. Molecular analysis of the gamma-glutamylcarboxylase gene revealed a heterozygous single nucleotide polymorphism, which decreases carboxylase activity and induces VK-dependent coagulation deficiency.
Assuntos
Carbono-Carbono Ligases/genética , DNA/genética , Polimorfismo Genético , Sangramento por Deficiência de Vitamina K/genética , Vitamina K/uso terapêutico , Antifibrinolíticos/uso terapêutico , Coagulação Sanguínea/genética , Carbono-Carbono Ligases/sangue , Feminino , Humanos , Recém-Nascido , Fatores de Risco , Índice de Gravidade de Doença , Sangramento por Deficiência de Vitamina K/tratamento farmacológico , Sangramento por Deficiência de Vitamina K/enzimologiaRESUMO
The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.